Characterization of GRP94-ligand interactions and purification, screening, and therapeutic methods relating thereto

ABSTRACT

The present invention discloses characterization of interactions between ligands and Hsp90 proteins, including GRP94, wherein ligand binding to the N-terminal nucleotide binding domain of GRP94 elicits a conformational change that converts the GRP94 from an inactive to an active conformation, and wherein the chaperone and peptide-binding activities of the GRP94 are markedly stimulated. Also disclosed are purification, screening, and therapeutic methods pertaining to the biological activity of GRP94, and in some instances HSP90, based upon the characterization of ligand interactions of Hsp90 peptide-binding proteins, including GRP94.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of International ApplicationNo. PCT/US01/09512 filed Mar. 26, 2001, which is based on and claimspriority to U.S. Provisional Application Serial No. 60/192,118, filedMar. 24, 2000. Both are herein incorporated by reference in theirentirety.

GRANT STATEMENT

[0002] This work was supported by NIH grant RO1 DK53058. Thus, the U.S.Government has certain rights in the invention.

TECHNICAL FIELD

[0003] The present invention relates to compositions and methodspertaining to the modulation of molecular chaperone function byregulatory ligands. In a preferred embodiment, the present inventionrelates to the characterization of ligand interactions of GRP94, andpurification, screening and therapeutic methods associated therewith.Table of Abbreviations 8-ANS 1,8-anilinonaphthalenesulfonate APC antigenpresenting cells BiP ER hsp70 homolog bis-ANS4,4′-dianilino-1,1-binaphthyl-5,5-disulfonic acid BMDC bonemarrow-derived dendritic cells BN-PAGE blue native polyacrylamide gelelectrophoresis CEA carcinoembryonic antigen(s) CT computed tomographicCTL cytotoxic T lymphocyte(s) DC dendritic cells DMEM Dulbecco'smodified Eagle's medium DTH delayed-type hypersensitivity ER endoplasmicreticulum GALT gut-associated lymphoid tissue GRP94 glucose regulatedprotein of 94 kDa, ER paralog of the Hsp90 family of chaperones HIVhuman immunodeficiency virus HPLC high pressure liquid chromatography hrhour(s) hsp(s) heat shock protein(s) HSP70 heat shock protein of 70 kDaHsp90 any member of the Hsp90 family of chaperones HSP90 heat shockprotein of 90 kDa HSV herpes simplex virus IFN interferon Igimmunoglobulin IGF-1 insulin-like growth factor IgG immunoglobulin G ILinterleukin MHC major histocompatability complex min minute MLTC mixedlymphocyte tumor cell assay NECA N-ethylcarboxamidoadenosine PDI proteindisulfide isomerase PSA prostate-specific antigen RSV respiratorysyncytial virus RT room temperature SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis TAP transporter associatedwith antigen presentation complex TFA trifluoroacetic acid TNF tumornecrosis factor

BACKGROUND ART

[0004] The pursuit of approaches for treatment and prevention of cancerand infectious diseases represents an ongoing effort in the medicalcommunity. Recent efforts to combat cancer and infectious disease haveincluded attempts to induce or enhance immune responses in subjectssuffering from a type of cancer or an infectious disease. See, e.g.Srivastava et al. (1998) Immunity 8:657-665.

[0005] Ischemia/reperfusion injury is a significant source of morbidityand mortality in a number of clinical disorders, including myocardialinfarction, cerebrovascular disease, and peripheral vascular disease. Inaddition, ischemia/reperfusion is relevant to the function oftransplanted organs and to the recovery expedience following anycardiovascular surgery. See Fan et al. (1999) J Mol Med 77:577-596.Thus, the identification of cellular protective mechanisms againstischemia-induced damage is a central goal for therapy of, for example,heart attacks, strokes, and neurodegenerative diseases, as well as forimprovement of recovery following surgery or transplantation.

[0006] The Hsp90 class of molecular chaperones are among the mostabundant proteins in eukaryotic cells. Hsp90 family members arephylogenetically ubiquitous whereas the endoplasmic reticulum paralog ofHSP90, GRP94 (gp96, ERp99, endoplasmin), is found only in higher plantsand metazoans (Nicchitta (1998) Curr Opin Immunol 10:103-109). The Hsp90family of proteins are known to be involved in directing the properfolding and trafficking of newly synthesized proteins and in conferringprotection to the cell during conditions of heat shock, oxidativestress, nutrient stress, and other physiological stress scenarios (Toft(1998) Trends Endocrinol Metab 9:238-243; Pratt (1998) Proc Soc Exp BiolMed 217:420-434). Under such stress conditions, protein folding, proteinoligomeric assembly, and protein stability can be profoundly disrupted.It is the function of the Hsp90 family of proteins, in concert withother molecular chaperones, to assist in preventing and reversingstress-induced inactivation of protein structure and function.

[0007] At a molecular level, HSP90 function in protein folding is knownto require the activity of a series of co-chaperones and accessorymolecules, including Hsp70 , p48Hip, p60Hop, p23, and FKBP52 (Prodromouet al. (1999) EMBO J 18:754-762; Johnson et al. (1996) J Steroid BiochemMol Biol 56:31-37; Chang et al. (1997) Mol Cell Biol 17:318-325; Duinaet al. (1996) Science 274:1713-1715; Chen et al. (1996) Mol Endocrinol10:682-693; Smith et al. (1993) J Biol Chem 268:18365-18371; Dittmar etal. (1998) J Biol Chem 273:7358-7366; Kosano et al. (1998) J Biol Chem273:3273-3279). These co-chaperones and accessory molecules participatein both concerted and sequential interactions with HSP90 and therebyserve to regulate its chaperone activity (Buchner (1999) Trends BiochemSci 24:136-141; Pratt et al. (1996) Exs 77:79-95; Pratt (1998) Proc SocExp Biol Med 217:420-434; Caplan (1999) Trends Cell Biol 9:262-268).

[0008] In addition to the contribution of co-chaperone proteins to theregulation of HSP90 function, recent crystallographic studies haveidentified an ATP/ADP binding pocket in the N-terminal domain of yeastand human HSP90, suggesting that HSP90 activity is regulated throughcyclic ATP binding and hydrolysis, as has been established for the Hsp70family of chaperones (Kassenbrock & Kelly (1989) EMBO J 8:1461-1467;Flynn et al. (1 989) Science 245:385-390; Palleros et al. (1 991) ProcNatl Acad Sci USA 88:519-523; Sriram et al. (1997) Structure 5:403-14;Prodromou et al. (1997) Cell 90:65-75; Obermann et al. (1998) J CellBiol 143:901-910; Csermely & Kahn (1991) J Biol Chem 266:4943-4950;Csermely et al. (1993) J Biol Chem 268:1901-1907; Sullivan et al. (1997)J Biol Chem 272:8007-8012; Scheibel et al. (1997) J Biol Chem272:18608-18613; Scheibel et al. (1998) Proc Natl Acad Sci USA95:1495-1499; Panaretou et al. (1998) EMBO J 17:4829-4836; Caplan (1999)Trends Cell Biol 9:262-268; Grenert et al. (1999) J Biol Chem274:17525-17533).

[0009] It has also been reported that HSP90 contains motifs bearingsignificant similarities to the Walker “A” and “B” sequences associatedwith ATP binding (Csermely & Kahn (1991) J Biol Chem 266:4943-4950;Jakob et al. (1996) J Biol Chem 271:10035-10041). Although thesesequences are substantially different from the consensus sequences foundamong serine and tyrosine kinases, they are homologous to the ATPbinding sequence seen in the Hsp70 family of proteins (Csermely & Kahn(1991) J Biol Chem 266:4943-4950). Consistent with sequence predictions,ATP binding, autophosphorylation activity, and ATPase activity have allbeen demonstrated for HSP90, though these findings are not withoutcontroversy (Csermely & Kahn (1991) J Biol Chem 266:4943-4950; Nadeau etal. (1993) J Biol Chem 268:1479-1487, Jakob et al. (1996) J Biol Chem271:10035-10041; Grenert et al. (1999) J Biol Chem 274:17525-17533;Scheibel et al. (1997) J Biol Chem 272:18608-18613; Prodromou et al.(1997) Cell 90:65-75).

[0010] In part because of the very low affinity of HSP90 for ATP, a rolefor ATP in the regulation of HSP90 function remained under questionuntil crystallographic resolution of the N-terminal domain of yeast andhuman HSP90 in association with bound adenosine nucleotides (Prodromouet al. (1997) Cell 90:65-75; Obermann et al. (1998) J Cell Biol143:901-910). Aided by atomic scale structural insights, amino acidresidues critical for ATP binding and hydrolysis were subsequentlyidentified and analyzed (Prodromou et al. (1997) Cell 90:65-75;Panaretou et al. (1998) EMBO J 17:4829-4836; Obermann et al. (1998) JCell Biol 143:901-910). Thus, in the human HSP90, aspartate 93 (D128 forGRP94; D79 for yeast HSP90) provides a direct hydrogen bond interactionwith the N6 group of the purine moiety of the adenosine ring and isessential for ATP binding (Prodromou et al. (1997) Cell 90:65-75;Obermann et al. (1998) J Cell Biol 143:901-910). Glutamate 47 (E82 forGRP94; E33 for yeast HSP90) was postulated to play an importantcatalytic role in ATP hydrolysis, based both on its location relative tobound nucleotide and through comparison with the ATP binding domain ofE. coli DNA gyrase B (Prodromou et al. (1997) Cell 90:65-75; Obermann etal. (1998) J Cell Biol 143:901-910). In subsequent mutagenesis studiesof yeast HSP90, it was observed that the D79 mutant was deficient in ATPbinding and that E47 mutants were deficient in ATP hydrolysis activity(Obermann et al. (1998) J Cell Biol 143:901-910; Panaretou et al. (1998)EMBO J 17:4829-4836). As further evidence for a function of theseresidues in HSP90 activity, yeast containing either mutant form of HSP90were inviable (Obermann et al. (1998) J Cell Biol 143:901-910; Panaretouet al. (1998) EMBO J 17:4829-4836).

[0011] Progress in the development of Hsp90-based therapeutic and otherapplications has been impeded by a lack of characterization of ligandinteractions of Hsp90 proteins, including GRP94. Despite theabove-described characterization of ATP interaction with HSP90, evidencein support of intrinsic ATP binding and ATPase activities with respectto GRP94 is controversial and, as with HSP90, a clear consensusregarding the molecular basis of an adenosine nucleotide-mediatedregulation of GRP94-substrate interactions has yet to emerge (Jakob etal. (1996) J Biol Chem 271:10035-10041; Wearsch & Nicchitta (1997) JBiol Chem 272:5152-5156; Li and Srivastava (1993) EMBO J 12:3143-3151;Csermely et al. (1995) J Biol Chem 270:6381-6388; Csermely et al. (1998)Pharmacol Ther 79:129-168).

[0012] What is needed, then, is characterization of ligand interactionsat the ligand binding pocket of a HSP90 protein, in particular GRP94 andHSP90. To this end, the present invention discloses methods forassessing ligand-HSP-90 chaperone interactions. Using such methods, theactive and inactive structural conformations of GRP94 and HSP90 areherein disclosed, and the regulative capacity of several compounds toinduce the active or inactive conformation is also demonstrated. Thedisclosure herein also provides purification, screening, and therapeuticmethods pertaining to the biological activity of Hsp90 proteins. Thus,the present invention meets a long-standing need in the art for methodsand compositions that contribute to the understanding, diagnosis andtreatment of disorders related to Hsp90 protein function.

SUMMARY OF THE INVENTION

[0013] A method for purifying a complex comprising a GRP94 protein isdisclosed. The method comprises: (a) contacting a complex comprising aGRP94 protein with a binding agent that preferentially binds GRP94, thebinding agent immobilized to a solid phase support, to immobilize thecomplex to the solid phase support; (b) collecting the remaining sample;and (c) eluting the complex from the solid phase support to givepurified complex in the eluate. The present invention also provides acomplex obtained by performing the disclosed method.

[0014] A method for isolating an antigenic molecule associated with acomplex comprising a GRP94 protein is also disclosed. The methodcomprises: (a) contacting a complex comprising GRP94 and an antigenicmolecule with a binding agent that preferentially binds GRP94, thebinding agent immobilized to a solid phase support, to immobilize thecomplex to the solid phase support; (b) collecting the remaining sample;(c) eluting the complex from the solid phase support to give purifiedcomplex in the eluate; and isolating the antigenic molecule from theeluate. The present invention also provides an antigenic moleculeisolated according to the disclosed method.

[0015] A method for detecting a GRP94 complex in a sample suspected ofcontaining a GRP94 complex is also disclosed. The method comprises (a)contacting the sample with a binding agent that preferentially bindsGRP94 under conditions favorable to binding a complex comprising GRP94to the binding substance to form a second complex there between; and (b)detecting the second complex via a label conjugated to the bindingsubstance or via a labeled reagent that specifically binds to the secondcomplex subsequent to its formation.

[0016] A kit for detecting, isolating, or purifying a complex comprisinga GRP94 protein and an antigenic molecule is also disclosed.

[0017] A method of screening a candidate substance for an ability tomodulate the biological activity of a Hsp90 protein is also disclosed.The method comprises: (a) establishing a test sample comprising a GRP94protein and a ligand for a Hsp90 protein; (b) administering a candidatesubstance to the test sample; and (c) measuring the effect of thecandidate substance on binding of a Hsp90 protein and the ligand for aHsp90 protein in the test sample to thereby determine the ability of thecandidate substance to modulate biological activity of a Hsp90 protein.In the method, the Hsp90 protein can comprise a GRP94 protein and theligand can comprise bis-ANS.

[0018] Further disclosed is a method for identifying a candidatesubstance as an activator of the biological activity of a Hsp90 protein.The method comprises: (a) establishing a test sample comprising a Hsp90protein and a candidate substance, (b) administering 8-ANS to the testsample, (c) detecting a fluorescence signal produced by the 8-ANS, and(d) identifying the candidate substance as an activator of thebiological activity of a Hsp90 protein based upon an amount offluorescence signal produced by the 8-ANS as compared to a controlsample. Preferably, the Hsp90 protein comprises GRP94 or HSP90.

[0019] A method is also provided for identifying a candidate substanceas an inhibitor of the biological activity of a Hsp90 protein. Themethod comprises: (a) establishing a test sample to induce aconformational change to the Hsp90 protein, (b) heat-shocking the testsample to induce a conformational change to the Hsp90 protein, (c)administering 8-ANS to the test sample, (d) detecting a fluorescencesignal produced by binding of 8-ANS to the Hsp90, and (e) identifyingthe candidate substance as an inhibitor of the biological activity of aHsp90 protein based upon an amount of fluorescence signal produced bythe 8-ANS as compared to a control sample. Preferably, the Hsp90 proteincomprises GRP94 or HSP90.

[0020] A method of modulating biological activity of a Hsp90 protein isalso disclosed. The method comprises contacting an Hsp90 protein with aneffective amount of a Hsp90 protein activity-modulating substance tothereby modulate the biological activity of the Hsp90 protein.Preferably, the Hsp90 protein is GRP94 or HSP90. A pharmaceuticalcomposition comprising a therapeutically effective amount of a modulatorof a biological activity of a Hsp90 protein, and a pharmaceuticallyacceptable diluent or vehicle, is also disclosed. Preferably, the Hsp90protein is GRP94 or HSP90.

[0021] A method of treating a subject suffering from a disorder whereinmodulation of the biological activity of a Hsp90 protein is desirable isalso disclosed. The method comprises administering to the subject aneffective amount of a Hsp90 protein activity modulator, wherebymodulation of the biological activity of a Hsp90 protein in the subjectis accomplished. Preferably, the Hsp90 protein is GRP94 or HSP90. Hsp90biological activity (in a preferred embodiment—GRP94 biologicalactivity) that is modulated can comprise immunogenicity, proteintransport from the endoplasmic reticulum, recovery from stress andtissue injury arising from, for example, hypoxia/anoxia, nutrientdeprivation, or heat stress, or combinations thereof. The disorder to betreated can comprise a type of cancer; an infectious disease; a disorderassociated with impaired protein transport from the endoplasmicreticulum; a disease state, such as cancer, wherein it would be oftherapeutic benefit to inhibit or block the egress of proteins (e.g.,growth factor receptors) from the endoplasmic reticulum; a disorderassociated with ischemia; or combinations thereof. The method canfurther comprise administering to the subject a composition comprising atherapeutically or prophylactically effective amount of a purifiedcomplex, said complex comprising a Hsp90 protein bound to an antigenicmolecule specific to said disorder.

[0022] A method for preparing an immunogenic composition for inducing animmune response in a vertebrate subject is also disclosed. The methodcomprises: (a) harvesting from a eukaryotic cell an immunogenic complexcomprising an Hsp90 protein non-covalently bound to an antigenicmolecule, said complex, when administered to said vertebrate subjectbeing operative at initiating an immune response in said vertebratesubject, wherein said eukaryotic cell has been treated with anactivating ligand; and (b) combining said complex with pharmaceuticallyacceptable carrier. Preferably, the Hsp90 protein is GRP94 or HSP90. Theligand can comprise bis-ANS.

[0023] A method for preparing an immunogenic composition for inducing animmune response in a vertebrate subject is also disclosed. The methodcomprises: (a) reconstituting in vitro an antigenic molecule and anHsp90 protein molecule in the presence of a Hsp90 activating ligand tothereby produce an immunogenic complex comprising a Hsp90 proteinnon-covalently bound to an antigenic molecule, said complex, whenadministered to said vertebrate subject being operative at initiating animmune response in said vertebrate subject; and (b) combining saidcomplex with pharmaceutically acceptable carrier. Preferably, the Hsp90protein is GRP94 or HSP90, and the ligand comprises bis-ANS.

[0024] A method for preparing an immunogenic composition for inducing animmune response in a vertebrate subject is also disclosed. The methodcomprises: (a) sensitizing antigen presenting cells in vitro with acomplex comprising a Hsp90 protein non-covalently bound to an antigenicmolecule and with an activating ligand; and (b) combining said at leastone sensitized antigen presenting cell with pharmaceutically acceptablecarrier. Preferably, the Hsp90 protein is GRP94 or HSP90, and the ligandcomprises bis-ANS.

[0025] Accordingly, it is an object of the present invention to providenovel purification methods, novel screening methods, and noveltherapeutic methods pertaining to the biological activity of GRP94 andother Hsp90 proteins. The object is achieved in whole or in part by thepresent invention.

[0026] An object of the invention having been stated hereinabove, otherobjects will become evident as the description proceeds when taken inconnection with the accompanying Drawings and Laboratory Examples asbest described herein below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1A is a graph depicting Prodan binding to GRP94 independentof GRP94 structural state. Fluorescence emission wavelength scans of 0.5μM native or heat shocked (hs) GRP94 were performed following exposureto 5 μM Prodan for 30 minutes. Values represent the maximal fluorescencerelative to that occurring with an identical concentration of heatshocked GRP94. Experiments were conducted at excitation wavelengths of360 nm (Prodan). All spectra were background corrected.

[0028]FIG. 1B is a graph depicting 8-ANS binding to GRP94, anddependence of such binding on GRP94 structural state. Fluorescenceemission wavelength scans of 0.5 μM native or heat shocked (hs) GRP94were performed following exposure to 5 μM 8-ANS for 30 minutes. Valuesrepresent the maximal fluorescence relative to that occurring with anidentical concentration of heat shocked GRP94. Experiments wereconducted at excitation wavelengths of 372 nm (8-ANS). All spectra werebackground corrected.

[0029]FIG. 1C is a graph depicting bis-ANS binding to GRP94, anddependence of such binding on GRP94 structural state. Fluorescenceemission wavelength scans of 0.5 μM native or heat shocked (hs) GRP94were performed following exposure to 5 μM bis-ANS for 20 hours. Valuesrepresent the maximal fluorescence relative to that occurring with anidentical concentration of heat shocked GRP94. Experiments wereconducted at excitation wavelengths of 393 nm (bis-ANS). All spectrawere background corrected.

[0030]FIG. 1D is a graph depicting a time course of bis-ANS binding toGRP94. Values represent the maximal fluorescence relative to thatoccurring with an identical concentration of heat shocked GRP94.Experiments were conducted at excitation wavelengths of 393 nm(bis-ANS). All spectra were background corrected.

[0031]FIG. 2A is a graph depicting kinetic analysis of bis-ANSinteractions with heat shocked GRP94. The concentration dependence ofbis-ANS binding to heat shocked GRP94 was conducted under experimentalconditions of fixed bis-ANS concentration (50 nM) and increasing GRP94concentration, as indicated.

[0032]FIG. 2B is a Klotz plot representation of bis-ANS/GRP94 bindingdata. Half maximal binding occurs at 110 nM GRP94. Excitation wavelenth,393 nm. Emission wavelength, 475 nm.

[0033]FIG. 3 is a digital image of a Coomassie Blue stained geldepicting that bis-ANS and heat shock increase GRP94 proteolysissensitivity. GRP94 (5 μg, 5 μM) was incubated with 50 μM bis-ANS for onehour at 37° C. or heat shocked for 15 minutes at 50° C. Samples werethen digested with 0.1% trypsin for 30 minutes at 37° C. and analyzed on12.5% SDS-PAGE gels. Lane 1, 5 μg of undigested GRP94; lane 2, controlnative GRP94 incubated with trypsin; lane 3, bis-ANS treated GRP94digested with trypsin; lane 4, GRP94 heat shocked then digested withtrypsin.

[0034]FIG. 4 is a digital image of a Coomassie Blue stained geldepicting that bis-ANS and heat shock induce GRP94 multimerization.GRP94 was heat shocked at 50° C. for 0-15 minutes or incubated with10-fold molar excess of bis-ANS and the structural state of the proteinanalyzed on 5-18% native blue polyacrylamide gradient gels. Themobilities of GRP94 dimers, tetramers, hexamers, and octamers are shown.Molecular weight standards are indicated to the right of FIG. 4.

[0035]FIG. 5 is a graph depicting that circular dichroism spectra ofnative, heat shocked, and bis-ANS treated GRP94 are identical. Circulardichroism spectra of 1 μM GRP94 native (diamonds); heat shocked (dot anddash); and treated 2 hours with 10 μM bis-ANS (dotted) are shown.Spectra were collected as described in Examples 1-8 below.

[0036]FIG. 6A is a digital image of a Coomassie Blue stained geldepicting that radicicol blocks bis-ANS structural transitions. GRP94 (5μM) was preincubated for one hour at 37° C. with 0-500 μM radicicol andsubsequently incubated for one hour at 37° C. with 50 μM bis-ANS,trypsinized, and the trypsin digestion pattern analyzed by SDS-PAGE.

[0037]FIG. 6B is a graph depicting that radicicol blocks heat shock andbis-ANS binding. GRP94 (0.5 μM) was preincubated with 0-10 μM radicicolfor one hour, heat shocked, and subsequently incubated with 1 μMbis-ANS. Bis-ANS binding was determined by spectrofluorometry withbis-ANS binding to native GRP94 in the absence of radicicol shown forcomparison. Excitation 393 nm, emission 410-600 nm.

[0038]FIG. 7A is a graph depicting that bis-ANS and heat shock stimulateGRP94 chaperone activity. Citrate synthase enzyme was diluted to 0.15 μMinto buffer containing no GRP94, 1 μM native GRP94, heat shocked GRP94,or GRP94 which had been preincubated for two hours with 10 μM bis-ANS,and citrate synthase aggregation at 43° C. was monitored by lightscattering at 500 nm in a thermostatted spectrofluorometer.

[0039]FIG. 7B is a bar graph depicting that bis-ANS and heat shockstimulate GRP94 peptide binding activity. Native, heat shocked, orbis-ANS treated GRP94 were incubated with a 10-fold molar excess of¹²⁵I-VSV8 peptide for 30 minutes at 37° C. Free peptide was removed byspin column chromatography and bound radioactive peptide quantitated bygamma counting.

[0040]FIG. 8 is a bar graph depicting that GRP94 and Hsp90 exhibitdifferential ligand binding. NECA and ATP binding to GRP94 was performedin the presence of 20 nM [³H]-NECA (closed bars) or 50 μM [³²P]ATP(hatched bars) for 1 hour at 4° C. Bound versus free nucleotide wereseparated by vacuum filtration. PEI treated glass filters (S&S #32,Schleicher and Schuell of Keene, N.H.) were used for the NECA bindingassay while nitrocellulose filters (S&S BA85, Schleicher and Schuell ofKeene, N.H.) were used to measure ATP binding. The data presented areaverages of triplicate points and are corrected for nonspecific ligandbinding.

[0041]FIG. 9A is a Scatchard plot depicting characterization of NECAbinding to GRP94. GRP94 was incubated with increasing concentrations ofNECA for 1 hour at 4° C. as described in Materials and Methods. Boundversus free NECA were then separated by vacuum filtration with glassfilters pretreated in 0.3% PEI.

[0042]FIG. 9B is a saturation curve depicting characterization of NECAbinding to GRP94. The curve is plotted with respect to GRP94 dimerconcentration. The maximal binding stoichiometry is 1 molecule of NECAper molecule of GRP94 dimer.

[0043]FIG. 9C is a graph depicting stoichiometry of GRP94 binding toNECA (solid oval) and radicicol (solid rectangle). NECA and radicicolbinding to GRP94 was assayed by isothermal titration calorimetry. GRP94was present at a concentration of 5 μM. NECA titrations were performedwith a 152 μM NECA stock whereas radicicol titrations were performedwith a 115 μM stock. ITC data were collected as μcal/sec versus time andthe area under individual injection peaks, determined with theinstrument software, was plotted.

[0044]FIG. 10A is a graph depicting a competition assay for NECA by theHsp90 family inhibitors, geldanamycin (♦) and radicicol (▪). GRP94 wasincubated with 20 nM [³H]-NECA and increasing concentrations ofcompetitors for 1 hour at 4° C. Bound NECA was separated from free byvacuum filtration with glass filters pre-treated in 0.3% PEI. All datapoints represent the average of triplicates points minus background(nonspecific NECA binding in the absence of protein).

[0045]FIG. 10B is a graph depicting a competition assay for NECA by ATP(♦), ADP (▪), and AMP (▴). GRP94 was incubated with 20 nM 3H-NECA andincreasing concentrations of competitors for 1 hour at 4° C. Bound NECAwas separated from free by vacuum filtration with glass filterspre-treated in 0.3% PEI. All data points represent the average oftriplicate points minus background (nonspecific NECA binding in theabsence of protein).

[0046]FIG. 10C is a graph depicting a competition assay for NECA byadenosine (▴), and cAMP (▪). GRP94 was incubated with 20 nM [³H]-NECAand increasing concentrations of competitors for 1 hour at 4° C. BoundNECA was separated from free by vacuum filtration with glass filterspre-treated in 0.3% PEI. All data points represent the average oftriplicates points minus background (nonspecific NECA binding in theabsence of protein).

[0047]FIG. 11 is a bar graph depicting that ligand binding specificityof GRP94 to the adenosine base. GRP94 was incubated with 20 nM [³H]-NECAand competitors, all at 50 μM final concentration for 1 hour at 4° C.,and bound vs. free NECA was separated by vacuum filtration with glassfilters pretreated in 0.3% PEI.

[0048]FIG. 12 is a graph depicting that binding of ATP, ADP, and AMP toGRP94 is sensitive to Mg²⁺ concentration. GRP94 was incubated for 1 hourat 4° C. in 50 mM Tris, 20 nM [³H]-NECA and one of the followingconcentrations of competitor: 3.1×10⁻⁶ M ATP, 3.1×10⁻⁵ M ADP, 6×10⁻⁴ MAMP, or 3.1×10⁻⁵ M adenosine. Reactions were performed in the presenceof 10 mM Mg(OAc)₂ (hatched bars) or in the presence of nominal,endogenous magnesium (closed bars). Bound vs. free NECA was separated byvacuum filtration with glass filters pretreated in 0.3% PEI.

[0049]FIG. 13A is a bar graph depicting the effects of NECA on GRP94autophosphorylation. 25 μl reactions consisting of 1 μM GRP94 (closedbars), 0.15 mM γ-³²PATP (6000 cpm/pmol), 10 mM Mg(OAc)₂, and 50 mMK-Hepes, pH 7.4 ) were incubated for 1 hour at 37° C. One (1) unitcasein kinase II (hatched bars) was incubated in the above conditionswith the addition of 4 μM casein. Competitors were added to theappropriate samples with a final concentration of 180 μM NECA in 3.6%DMSO, 180 μM radicicol in 3.6% DMSO, 5 μg/ml heparin, 5 mM GTP, or 3.6%DMSO. Phosphorylated species were quantitated on a Fuji MACBAS1000™phosphorimaging system, and the average PSL units of three independentexperiments are displayed.

[0050]FIG. 13B is a bar graph depicting ATP hydrolysis in the presenceand absence of GRP94. 100 μl reactions consisting of 1 μM GRP94 monomer,various concentrations of MgATP (pH 7.0), and 50 mM K-Hepes, pH 7.4,were incubated for two hours at 37° C. ATP and ADP were separated on aHewlett Packard HPLC using a Partisil SAX column. Spontaneous ATPhydrolysis was determined in the absence of protein. Hydrolysis in thepresence of GRP94 is indicated by closed bars and spontaneous hydrolysisis indicated by the hatched bars.

[0051]FIG. 14 is a graph depicting ligand-induced conformational changesof GRP94. GRP94 (50 μg/ml) was incubated in buffer A supplemented with10 mM Mg(OAc)₂ and the following concentrations of ligands for 1 hour at37° C.: 50 μM NECA, 50 μM geldanamycin, 2.5 mM ATP, or 2.5 mM ADP.Samples were excited at a wavelength of 295 nm and the tryptophanemission spectra were recorded from 300-400 nm. All spectra werecorrected by subtraction of spectra obtained in buffer alone orbuffer+ligand samples.

DETAILED DESCRIPTION OF THE INVENTION

[0052] Disclosed herein is the characterization of ligand interactionsof GRP94, and where applicable Hsp90 , wherein ligand binding to theN-terminal nucleotide binding domain of GRP94, and in some instances,Hsp90, elicits a conformational change that converts GRP94, and in someinstances, Hsp90, from an inactive to an active conformation, andwherein the chaperone and peptide binding activities of GRP94, and whereapplicable, Hsp90, are markedly stimulated. Also disclosed herein is thecharacterization of ligand interactions of GRP94, and where applicableHsp90, wherein ligand binding to the N-terminal nucleotide bindingdomain of GRP94, and in some instances, Hsp90, inhibits a conformationalchange that converts GRP94, and in some instances, Hsp90, from aninactive to an active conformation, and wherein the activities of GRP94,and where applicable, Hsp90, are markedly inhibited. Also disclosedherein are ligands, and methods of screening for such ligands, that bindto the N-terminal nucleotide binding domain and inhibit protein activityand/or protein conformational activation in a manner similar and/orrelated to that observed with geldanamycin and radicicol. Such ligandscan function as potential anti-tumor therapeutics. Also disclosed hereinare purification, screening, and therapeutic methods pertaining to thebiological activity of GRP94, and in some instances Hsp90, based uponthe characterization of ligand interactions of GRP94, and in someinstances Hsp90.

[0053] A. Definitions

[0054] While the following terms are believed to have well definedmeanings in the art, the following definitions are set forth tofacilitate explanation of the invention.

[0055] “Antigenic molecule” as used herein refers to the peptides withwhich GRP94 or HSP90 endogenously associates in vivo (e.g., in infectedcells or precancerous or cancerous tissue) as well as exogenousantigens/immunogens (i.e., not complexed with GRP94 or HSP90 in vivo) orantigenic/immunogenic fragments and derivatives thereof.

[0056] The term “biological activity” is meant to refer to a moleculehaving a biological or physiological effect in a subject. Adjuvantactivity is an example of a biological activity. Activating or inducingproduction of other biological molecules having adjuvant activity isalso a contemplated biological activity.

[0057] The term “adjuvant activity” is meant to refer to a moleculehaving the ability to enhance or otherwise modulate the response of avertebrate subject's immune system to an antigen.

[0058] The term “immune system” includes all the cells, tissues,systems, structures and processes, including non-specific and specificcategories, that provide a defense against antigenic molecules,including potential pathogens, in a vertebrate subject. As is well knownin the art, the non-specific immune system includes phagocytic cellssuch as neutrophils, monocytes, tissue macrophages, Kupffer cells,alveolar macrophages, dendritic cells and microglia. The specific immunesystem refers to the cells and other structures that impart specificimmunity within a host. Included among these cells are the lymphocytes,particularly the B cell lymphocytes and the T cell lymphocytes. Thesecells also include natural killer (NK) cells. Additionally,antibody-producing cells, like B lymphocytes, and the antibodiesproduced by the antibody-producing cells are also included within theterm “immune system”.

[0059] The term “immune response” is meant to refer to any response toan antigen or antigenic determinant by the immune system of a vertebratesubject. Exemplary immune responses include humoral immune responses(e.g. production of antigen-specific antibodies) and cell-mediatedimmune responses (e.g. lymphocyte proliferation), as defined hereinbelow.

[0060] The term “systemic immune response” is meant to refer to animmune response in the lymph node-, spleen-, or gut-associated lymphoidtissues wherein cells, such as B lymphocytes, of the immune system aredeveloped. For example, a systemic immune response can comprise theproduction of serum IgG's. Further, systemic immune response refers toantigen-specific antibodies circulating in the blood stream andantigen-specific cells in lymphoid tissue in systemic compartments suchas the spleen and lymph nodes.

[0061] The terms “humoral immunity” or “humoral immune response” aremeant to refer to the form of acquired immunity in which antibodymolecules are secreted in response to antigenic stimulation.

[0062] The terms “cell-mediated immunity” and “cell-mediated immuneresponse” are meant to refer to the immunological defense provided bylymphocytes, such as that defense provided by T cell lymphocytes whenthey come into close proximity to their victim cells. A cell-mediatedimmune response also comprises lymphocyte proliferation. When“lymphocyte proliferation” is measured, the ability of lymphocytes toproliferate in response to specific antigen is measured. Lymphocyteproliferation is meant to refer to B cell, T-helper cell or CTL cellproliferation.

[0063] The term “CTL response” is meant to refer to the ability of anantigen-specific cell to lyse and kill a cell expressing the specificantigen. As described herein below, standard, art-recognized CTL assaysare performed to measure CTL activity.

[0064] “Adoptive immunotherapy” as used herein refers to a therapeuticapproach with particular applicability to cancer whereby immune cellswith an antitumor reactivity are administered to a tumor-bearing host,with the aim that the cells mediate either directly or indirectly, theregression of an established tumor.

[0065] An “immunogenic composition” is meant to refer to a compositionthat can elicit an immune response. A vaccine is contemplated to fallwithin the meaning of the term “immunogenic composition”, in accordancewith the present invention.

[0066] The term “a biological response modifier” is meant to refer to amolecule having the ability to enhance or otherwise modulate a subject'sresponse to a particular stimulus, such as presentation of an antigen.

[0067] As used herein, the terms “candidate substance” and “candidatecompound” are used interchangeably and refer to a substance that isbelieved to interact with another moiety as a biological responsemodifier. For example, a representative candidate compound is believedto interact with a complete, Hsp90 protein, or fragment thereof, andwhich can be subsequently evaluated for such an interaction. Exemplarycandidate compounds that can be investigated using the methods of thepresent invention include, but are not restricted to, agonists andantagonists of a Hsp90 protein, viral epitopes, peptides, enzymes,enzyme substrates, co-factors, lectins, sugars, oligonucleotides ornucleic acids, oligosaccharides, proteins, chemical compounds smallmolecules, and monoclonal antibodies.

[0068] As used herein, the term “modulate” means an increase, decrease,or other alteration of any or all chemical and biological activities orproperties of a wild-type or mutant Hsp90 protein, preferably awild-type or mutant GRP94 or HSP90 polypeptide. The term “modulation” asused herein refers to both upregulation (i.e., activation orstimulation) and downregulation (i.e. inhibition or suppression) of aresponse.

[0069] As used herein, the term “agonist” means an agent thatsupplements or potentiates the biological activity of a functional Hsp90protein.

[0070] As used herein, the term “antagonist” means an agent thatdecreases or inhibits the biological activity of a functional Hsp90protein, or that supplements or potentiates the biological activity of anaturally occurring or engineered non-functional Hsp90 protein.

[0071] B. General Considerations

[0072] As used herein the term “Hsp90 protein” is meant to refer to anyof the Hsp90 class of molecular chaperones that are among the mostabundant proteins in eukaryotic cells, and to biologically activefragments of such proteins. The term “HSP90 protein” refers to anindividual member of this class, exemplified by canine HSP90 (GenBankAccession No. U01153) and mouse HSP90 (SwissProt Accession No. P08113),and to biologically active fragments thereof. Hsp90 family members arephylogenetically ubiquitous whereas the endoplasmic reticulum paralog ofHSP90, GRP94 (gp96, ERp99, endoplasmin) is found only in higher plantsand metazoans (Nicchitta (1998) Curr Opin Immunol 10:103-109). The Hsp90family of proteins are involved in directing the proper folding andtrafficking of newly synthesized proteins and in conferring protectionto the cell during conditions of heat shock, oxidative stress,hypoxic/anoxic conditions, nutrient deprivation, other physiologicalstresses, and disorders or traumas that promote such stress conditionssuch as, for example, stroke and myocardial infarction.

[0073] As used herein, the terms “binding pocket of the Hsp90 protein”,“Hsp90 binding pocket”, “GRP94 binding pocket”, and “HSP90 bindingpocket” are used interchangeably and mean that region of a Hsp90protein, preferably a GRP94 polypeptide or a HSP90 polypeptide, where aligand binds. Even more preferably, the GRP94 binding pocket comprisesamino acid residues 22-337 of GRP94.

[0074] As noted above, GRP94 (gp96, ERp99, endoplasmin) is theendoplasmic reticulum paralog of cytosolic HSP90, and as such, is anabundant resident ER lumenal protein that by virtue of its associationwith nascent polypeptides performs a chaperone function. The term“GRP94” and/or “GRP94 protein” also refers to biologically activefragments of a GRP94 protein. Consistent with this role, GRP94expression is upregulated by stress conditions that promote proteinmisfolding or unfolding, such as glucose starvation, oxidative stress,and heavy metal poisoning. In addition to its role in the regulation ofprotein folding in the ER, GRP94 can function in the intercellulartrafficking of peptides from the extracellular space to the majorhistocompatability complex (MHC) class I antigen processing pathway ofprofessional antigen presenting cells. Thus, in addition to ahomeostatic role in protein folding and assembly, GRP94 functions as acomponent of the MHC class I antigen processing and presentationpathways of mammalian cells.

[0075] GRP94 also contributes to the folding and assembly ofimmunoglobulins, MHC class II molecules, HSV-1 glycoproteins,thyroglobulin, collagen, and p185erbB2. (Melnick et al. (1992) J BiolChem 267:21303-21306; Melnick et al. (1994) Nature 370:373-375; Schaiffet al. (1992) J Exp Med 176:657-666; Navarro et al. (1991) Virology184:253-264; Kuznetsov et al. (1994) J Biol Chem 269:22990-22995;Ferreira et al. (1994) J Cell Biochem 56:518-26; Chavany et al. (1996) JBiol Chem 273:4974-4977). In addition to interactions with polypeptidefolding substrates, GRP94 binds peptides, a subset of which is suitablefor assembly on nascent MHC class I molecules. (Srivastava et al. (1986)Proc Natl Acad Sci USA 83:3407-3411; Nieland et al. (1996) Proc NatlAcad Sci USA 93:6135-6139; Wearsch & Nicchitta (1997) J Biol Chem272:5152-5156; Ishii et al. (1999) J Immunol 162:1303-1309; Srivastavaet al. (1998) Immunity 8:657-665; Sastry & Linderoth (1999) J Biol Chem274:12023-12035). The peptide binding activity of GRP94 plays a role inits ability to elicit CD8⁺ T cell immune responses. (Udono et al. (1994)Proc Natl Acad Sci USA, 91:3077-30781; Suto & Srivastava (1995) Science269:1585-1588; Arnold et al. (1995) J Exp Med 182:885-889; Nair et al.(1999) J Immunol 162:6426-6432; Blachere et al. (1997) J Exp Med186:465-472; Heike et al. (1996) J Leukoc Biol 139:613-623; Srivastavaet al. (1998) Immunity 8:657-665). Peptide binding activity is not,however, alone sufficient to impart immunogenic activity to a proteinand thus GRP94 is among a limited subset of molecular chaperones thatcan function in the essential immunological process ofcross-presentation. (Srivastava et al. (1998) Immunity 8:657-665; Nairet al. (1999) J Immunol 162:6426-6432; Basu and Srivastava (1999) J ExpMed 189:797-802; Schild et al. (1999) Curr Opin Immunol 11:109-113).

[0076] HSP90 has adenosine nucleotide-dependent modes of regulation.Additionally, amino acid side chains that participate in water-mediatedhydrogen bonds with the N7 group of the purine ring of adenosine (N51 inhuman HSP90=N86 in GRP94) and the N1 group of the purine ring ofadenosine (G97 in human HSP90=G130 of GRP94) are conserved between HSP90and GRP94. The N6 group of the purine ring of adenosine (L48 in humanHSP90=L83 in GRP94) that mediates direct nucleotide binding is alsoconserved between HSP90 and GRP94. In ATP binding with HSP90, the N6group of the adenine purine is the sole direct hydrogen bond between thenucleotide and the nucleotide binding pocket (Prodromou et al. (1997)Cell 90:65-75; Obermann et al. (1998) J Cell Biol 143:901-910), and N6substituted adenosine analogs do not bind to GRP94. (Hutchison & Fox(1989) J Biol Chem 264:19898-903; Hutchison et al. (1990) Biochemistry29:5138-5144). Thus, although ATP/ADP binding and hydrolysis aregenerally accepted as biological properties of HSP90, it is not knownwhether ATP/ADP serve an identical function(s) in the regulation ofGRP94 activity. ATP and ADP bind with very low affinity to GRP94 andthus experimental limitations require that ATP/ADP interactions at theGRP94 nucleotide binding pocket be analyzed by indirect methods,including but not limited to ligand displacement assays. (Wearsch et al.(1998) Biochemistry 37(16):5709-5719; Csermely et al. (1995) J Biol Chem270:6381-6388; Li & Srivastava (1993) EMBO J 12:3143-3151).

[0077] The peptide binding activity of GRP94 plays a role in its abilityto elicit CD8⁺ T cell immune responses. Peptide binding activity is not,however, alone sufficient to impart immunogenic activity to a proteinand thus GRP94 is among a limited subset of molecular chaperones thatcan function in the essential immunological process ofcross-presentation. Until the disclosure of the present invention, aGRP94 ligand-interaction that modulates activity of GRP94 with respectto both polypeptide and peptide substrates remained to be determined.

[0078] HSP90 and GRP94 have been proposed as possible targets of severalantitumor agents, principally radicicol and geldanamycin. Scheibel &Buckner (1998) Biochem Pharm 56:675-82. These compounds are believed toact by inhibiting the ability of the Hsp90 proteins to assistproto-oncogenic kinases, hormone receptors, and other signaling proteinsassume their active folded states and appropriate subcellular location.Pratt (1998) Proc Soc Exp Biol Med 217:420-434.

[0079] GRP94 has also been found to elicit cytotoxic T cell responses, areflection of its peptide binding activity (Nicchitta (1998) Curr OpinImmunol 10:103-109; Srivastava et al. (1998) Immunity 8:657-665). It isnow established that GRP94-peptide complexes can be processed byprofessional antigen presenting cells, with the GRP94-bound peptidesexchanged onto MHC class I molecules of the antigen presenting cell. Theantigen presenting cells can then interact with naive CD8⁺ T cellresponses against tissue(s) displaying peptide epitopes present incomplex with GRP94 (Srivastava et al. (1998) Immunity 8:657-665).

[0080] A potential yet heretofore uncharacterized protective role ofgrp94 in inschemia is supported by the observation that expression ofGRP94 is enhanced in hippocampus after transient forebrain ischemia of aduration known to result in neuronal death (Yagita et al. (1999) JNeurochem 72:1544-1551). grp94 is similarly induced following acutekidney ischemia (Kuznetsov (1996) Proc Natl Acad Sci USA 93:8584-8589).Heat-shock proteins, including HSP90, are overexpressed during theoxidative stress of reperfusion that generally follows ischemia(Sciandra et al. (1984) Proc Natl Acad Sci USA 81:4843-4847). HSP90might also play a role in ischemic signaling by binding to thehypoxia-inducible factor 1-a (Gradin et al. (1996) Mol Cell Biol16:5221-5231).

[0081] Summarily, in accordance with the present invention, GRP94 andHSP90 represent rational targets for chemotherapeutics,immunotherapeutics and vaccines relevant to the treatment of infectionsdisease and cancer. In view of their function as molecular chaperones,GRP94 and HSP90 further represent rational targets for the developmentof therapeutics for tissue injury and stress, such as may occur inischemic injuries including, but not limited to, organ (kidney, heart,lung, liver) transplantation, cerebral stroke, and myocardial infarct.Furthermore, Hsp90 and GRP94 represent rational targets for anti-tumordrug design.

[0082] C. Ligand Compositions

[0083] In one embodiment the present invention pertains to a compositionof matter that acts as a ligand for GRP94. The ligand can comprise apurified and isolated natural ligand for GRP94, or can comprise asynthetic compound, such as are identified by the screening and rationaldrug design techniques disclosed herein. Preferably, the ligand is asmall molecule mimetic. More preferably, the ligand has activity in themodulation of GRP94 biological activity. Thus, ligands having suchactivity are also referred to herein as “modulators”. Representativeligand compositions are preferably about 500-1000 daltons, polycyclicmolecules that can show structural resemblance to radicicol,geldanamycin, or adenosine derivatives. Optionally, a ligand ishydrophobic.

[0084] A representative ligand or modulator composition of mattercomprises an adenosine moiety or structural mimetic thereof having anyof a variety of substitutions at the 2′, 3′, and 5′ positions, in thecase of adenosine, as deemed appropriate by high resolution structuralanalyses of ligand-GRP94 interactions. Optionally, 5′ position alkylextensions can be included, preferably as a carboxamido linkage to theparent adenosine and, to facilitate stable chemical linkage to a solidsupport for the purposes of affinity-based purification, terminating inany of a subset of chemically reactive groups including, but not limitedto vinyl, maleimide and/or succinimide esters, or substituents suitablefor chemical coupling to solid phase supports, such as amino orsulphydryl groups. The composition acts as a ligand for GRP94 and hasapplication in the purification, screening and therapeutic methodsdisclosed herein.

[0085] Additional ligands can be identified through combinatorialchemistry of a parent precursor molecule bearing a hydrogen bondmimetic, preferably corresponding to the ribose of adenosine, and abenzimidazole or structurally related scaffold, corresponding to theadenine base of adenosine.

[0086] A representative ligand or modulator composition comprises acompound of the formula (I):

[0087] where:

[0088] X and Y are the same or different and X and Y═C, N, O or S; and Xand Y can be substituted with hydrogen, hydroxyl, or oxygen, includingdouble-bonded oxygen;

[0089] R¹=hydrogen, hydroxyl, C₁ to C₆ alkyl, C₁ to C₆ branched alkyl,C₁ to C₆ hydroxyalkyl, branched C₁ to C₆ hydroxyalkyl, C₄ to C₈cycloalkyl, C₁ to C₆ alkenyl, branched C₁ to C₆ alkenyl, C₄ to C₈cycloalkenyl, C₄ to C₈ aryl, C₄ to C₈ aroyl, C₄ to C₈ aryl-substitutedC₁ to C₆ alkyl, C₁ to C₆ alkoxy, C₁ to C₆ branched alkoxy, C₄ to C₈aryloxy, primary, secondary or tertiary C₁ to C₆ alkylamino, primary,secondary or tertiary branched C₁ to C₆ alkylamino, primary, secondaryor tertiary cycloalkylamino, primary, secondary or tertiary C₄ to C₈arylamino, C₁ to C₆ alkylcarboxylic acid, branched C₁ to C₆alkylcarboxylic acid, C₁ to C₆ alkylester, branched C₁ to C₆ alkylester,C₄ to C₈ arylcarboxylic acid, C₄ to C₈ arlyester, C₄ to C₈ arylsubstituted C₁ to C₆ alkyl, C₄ to C₁₂ heterocyclic or heteropolycyclicalkyl or aryl with O, N or S in the ring, alkyl-substituted oraryl-substituted C₄ to C₁₂ heterocyclic or heteropolycyclic alkyl oraryl with O, N or S in the ring; or hydroxyl-, amino-, orhalo-substituted versions thereof; or R¹ is halo where halo is chloro,fluoro, bromo, or iodo;

[0090] R²=hydrogen, hydroxyl, C₁ to C₆ alkyl, C₁ to C₆ branched alkyl,C₁ to C₆ hydroxyalkyl, branched C₁ to C₆ hydroxyalkyl, C₄ to C₈cycloalkyl, C₁ to C₆ alkenyl, branched C₁ to C₆ alkenyl, C₄ to C₈cycloalkenyl, C₄ to C₈ aryl, C₄ to C₈ aroyl, C₄ to C₈ aryl-substitutedC₁ to C₆ alkyl, C₁ to C₆ alkoxy, C₁ to C₆ branched alkoxy, C₄ to C₈aryloxy, primary, secondary or tertiary C₁ to C₆ alkylamino, primary,secondary or tertiary branched C₁ to C₆ alkylamino, primary, secondaryor tertiary cycloalkylamino, primary, secondary or tertiary C₄ to C₈arylamino, C₁ to C₆ alkylcarboxylic acid, branched C₁ to C₆alkylcarboxylic acid, C₁ to C₆ alkylester, branched C₁ to C₆ alkylester,C₄ to C₈ arylcarboxylic acid, C₄ to C₈ arlyester, C₄ to C₈ arylsubstituted C₁ to C₆ alkyl, C₄ to C₁₂ heterocyclic or heteropolycyclicalkyl or aryl with O, N or S in the ring, alkyl-substituted oraryl-substituted C₄ to C₁₂ heterocyclic or heteropolycyclic alkyl oraryl with O, N or S in the ring; or hydroxyl-, amino-, orhalo-substituted versions thereof; or R² is halo where halo is chloro,fluoro, bromo, or iodo; and

[0091] R³=hydrogen, hydroxyl, C₁ to C₆ alkyl, C₁ to C₆ branched alkyl,C₁ to C₆ hydroxyalkyl, branched C₁ to C₆ hydroxyalkyl, C₄ to C₈cycloalkyl, C₁ to C₆ alkenyl, branched C₁ to C₆ alkenyl, C₄ to C₈cycloalkenyl, C₄ to C₈ aryl, C₄ to C₈ aroyl, C₄ to C₈ aryl-substitutedC₁ to C₆ alkyl, C₁ to C₆ alkoxy, C₁ to C₆ branched alkoxy, C₄ to C₈aryloxy, primary, secondary or tertiary C₁ to C₆ alkylamino, primary,secondary or tertiary branched C₁ to C₆ alkylamino, primary, secondaryor tertiary cycloalkylamino, primary, secondary or tertiary C₄ to C₈arylamino, C₁ to C₆ alkylcarboxylic acid, branched C₁ to C₆alkylcarboxylic acid, C₁ to C₆ alkylester, branched C₁ to C₆ alkylester,C₄ to C₈ arylcarboxylic acid, C₄ to C₈ arlyester, C₄ to C₈ arylsubstituted C₁ to C₆ alkyl, C₄ to C₁₂ heterocyclic or heteropolycyclicalkyl or aryl with O, N or S in the ring, alkyl-substituted oraryl-substituted C₄ to C₁₂ heterocyclic or heteropolycyclic alkyl oraryl with O, N or S in the ring; or hydroxyl-, amino-, orhalo-substituted versions thereof; or R³ is halo where halo is chloro,fluoro, bromo, or iodo.

[0092] Where the ligand composition further comprises a compound of theformula (II):

[0093] where:

[0094] X and Y are the same or different and X and Y═C, N, O or S; and Xand Y can be substituted with hydrogen, hydroxyl, or oxygen, includingdouble-bonded oxygen;

[0095] R¹=hydrogen, hydroxyl, C₁ to C₆ alkyl, C₁ to C₆ branched alkyl,C₁ to C₆ hydroxyalkyl, branched C₁ to C₆ hydroxyalkyl, C₄ to C₈cycloalkyl, C₁ to C₆ alkenyl, branched C₁ to C₆ alkenyl, C₄ to C₈cycloalkenyl, C₄ to C₈ aryl, C₄ to C₈ aroyl, C₄ to C₈ aryl-substitutedC₁ to C₆ alkyl, C₁ to C₆ alkoxy, C₁ to C₆ branched alkoxy, C₄ to C₈aryloxy, primary, secondary or tertiary C₁ to C₆ alkylamino, primary,secondary or tertiary branched C₁ to C₆ alkylamino, primary, secondaryor tertiary cycloalkylamino, primary, secondary or tertiary C₄ to C₈arylamino, C₁ to C₆ alkylcarboxylic acid, branched C₁ to C₆alkylcarboxylic acid, C₁ to C₆ alkylester, branched C₁ to C₆ alkylester,C₄ to C₈ arylcarboxylic acid, C₄ to C₈ arlyester, C₄ to C₈ arylsubstituted C₁ to C₆ alkyl, C₄ to C₁₂ heterocyclic or heteropolycyclicalkyl or aryl with O, N or S in the ring, alkyl-substituted oraryl-substituted C₄ to C₁₂ heterocyclic or heteropolycyclic alkyl oraryl with O, N or S in the ring; or hydroxyl-, amino-, orhalo-substituted versions thereof; or R¹ is halo where halo is chloro,fluoro, bromo, or iodo;

[0096] R²=hydrogen, hydroxyl, C₁ to C₆ alkyl, C₁ to C₆ branched alkyl,C₁ to C₆ hydroxyalkyl, branched C₁ to C₆ hydroxyalkyl, C₄ to C₈cycloalkyl, C₁ to C₆ alkenyl, branched C₁ to C₆ alkenyl, C₄ to C₈cycloalkenyl, C₄ to C₈ aryl, C₄ to C₈ aroyl, C₄ to C₈ aryl-substitutedC₁ to C₆ alkyl, C₁ to C₆ alkoxy, C₁ to C₆ branched alkoxy, C₄ to C₈aryloxy, primary, secondary or tertiary C₁ to C₆ alkylamino, primary,secondary or tertiary branched C₁ to C₆ alkylamino, primary, secondaryor tertiary cycloalkylamino, primary, secondary or tertiary C₄ to C₈arylamino, C₁ to C₆ alkylcarboxylic acid, branched C₁ to C₆alkylcarboxylic acid, C₁ to C₆ alkylester, branched C₁ to C₆ alkylester,C₄ to C₈ arylcarboxylic acid, C₄ to C₈ arlyester, C₄ to C₈ arylsubstituted C₁ to C₆ alkyl, C₄ to C₁₂ heterocyclic or heteropolycyclicalkyl or aryl with O, N or S in the ring, alkyl-substituted oraryl-substituted C₄ to C₁₂ heterocyclic or heteropolycyclic alkyl oraryl with O, N or S in the ring; or hydroxyl-, amino-, orhalo-substituted versions thereof; or R² is halo where halo is chloro,fluoro, bromo, or iodo;

[0097] R³=hydrogen, hydroxyl, C₁ to C₆ alkyl, C₁ to C₆ branched alkyl,C₁ to C₆ hydroxyalkyl, branched C₁ to C₆ hydroxyalkyl, C₄ to C₈cycloalkyl, C₁ to C₆ alkenyl, branched C₁ to C₆ alkenyl, C₄ to C₈cycloalkenyl, C₄ to C₈ aryl, C₄ to C₈ aroyl, C₄ to C₈ aryl-substitutedC₁ to C₆ alkyl, C₁ to C₆ alkoxy, C₁ to C₆ branched alkoxy, C₄ to C₈aryloxy, primary, secondary or tertiary C₁ to C₆ alkylamino, primary,secondary or tertiary branched C₁ to C₆ alkylamino, primary, secondaryor tertiary cycloalkylamino, primary, secondary or tertiary C₄ to C₈arylamino, C₁ to C₆ alkylcarboxylic acid, branched C₁ to C₆alkylcarboxylic acid, C₁ to C₆ alkylester, branched C₁ to C₆ alkylester,C₄ to C₈ arylcarboxylic acid, C₄ to C₈ arlyester, C₄ to C₈ arylsubstituted C₁ to C₆ alkyl, C₄ to C₁₂ heterocyclic or heteropolycyclicalkyl or aryl with O, N or S in the ring, alkyl-substituted oraryl-substituted C₄ to C₁₂ heterocyclic or heteropolycyclic alkyl oraryl with O, N or S in the ring; or hydroxyl-, amino-, orhalo-substituted versions thereof; or R³ is halo where halo is chloro,fluoro, bromo, or iodo; and

[0098] R⁴=C₁ to C₆ alkyl, C₁ to C₆ branched alkyl, C₄ to C₈ cycloalkylwith or without O, N or S in the ring, C₁ to C₆ alkenyl, branched C₁ toC₆ alkenyl, C₄ to C₈ cycloalkenyl with or without O, N or S in the ring,C₄ to C₈ aroyl, C₄ to C₈ aryl, C₄ to C₁₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring, C₄ to C₈aryl-substituted C₁ to C₆ alkyl, alkyl-substituted or aryl-substitutedC₄ to C₁₂ heterocyclic or heteropolycyclic alkyl or aryl with O, N or Sin the ring, alkyl-substituted C₄ to C₈ aroyl, or alkyl-substituted C₄to C₈ aryl; or hydroxyl-, amino-, or halo-substituted versions thereofwhere halo is chloro, bromo, fluoro or iodo.

[0099] D. Purification Methods

[0100] In accordance with the present invention, a method for purifyinga complex comprising GRP94, or in some instances HSP90, by affinitychromatography is provided. The complex preferably comprises GRP94 boundto an antigenic molecule. More preferably, the complex comprises GRP94non-covalently bound to an antigenic molecule. In one embodiment, themethod comprises contacting a sample comprising a GRP94 complex with abinding agent that preferentially binds GRP94, the binding agentimmobilized to a solid phase support, to immobilize the complex to thesolid phase support; collecting the remaining sample; and eluting theGRP94 complex from the solid phase support to give purified GRP94complex in the eluate. By the phrase “a binding agent thatpreferentially binds GRP94” it is meant an agent that preferentiallybinds GRP94 as compared to other molecular entities, including but notlimited to other heat shock proteins.

[0101] The binding agent preferably comprises an adenosine moiety orstructural mimetic thereof having any of a variety of substitutions atthe 2′, 3′, and 5′ positions, in the case of adenosine, as deemedappropriate by high resolution structural analyses of ligand-GRP94interactions. Optionally, 5′ position alkyl extensions can be included,preferably as a carboxamido linkage to the parent adenosine and, tofacilitate stable chemical linkage to a solid support for the purposesof affinity-based purification, terminating in any of a subset ofchemically reactive groups including, but not limited to vinyl,maleimide and/or succinimide esters, or substituents suitable forchemical coupling to solid phase supports, such as amino or sulphydrylgroups. More preferably, the binding agent is free of ATP or ADP. Arepresentative binding agent comprises a compound of the formula (I) ora compound of formula (II). Another representative binding agentcomprises N-ethylcarboxamidoadenosine (NECA). Additional ligands can beidentified through combinatorial chemistry of a parent precursormolecule bearing a hydrogen bond mimetic, preferably corresponding tothe ribose of adenosine, and a benzimidazole or structurally relatedscaffold, corresponding to the adenine base of adenosine.

[0102] Optionally, the complex bound to the immobilized binding agent iseluted by washing the solid phase support with a buffer comprising aphysiological salts solution containing appropriate concentrations ofthe parent ligand (i.e., the binding agent) to give complex in theeluate. Hence, a complex further comprising the binding agent or elutingligand is also provided in accordance with the present invention. Theeluting ligand will then be removed from the eluate solution by dialysisin buffers appropriate for GMP production including, but not limited to,physiological salts and volatile salts.

[0103] The affinity methods disclosed herein above can be used toisolate GRP94-peptide complexes or GRP94 alone, or in some instances,HSP90-peptide complexes, or the HSP90 protein alone, from any eukaryoticcell. For example, tissues, isolated cells, or immortalized eukaryotecell lines infected with a preselected intracellular pathogen, tumorcells or tumor cell lines can be used. The complex can also be obtainedfrom a vertebrate subject, such as a warm-blooded vertebrate, includingmammals and bird. Optionally, the mammal includes, but is not limitedto, human, mouse, pig, rat, ape, monkey, cat, guinea pig, cow, goat andhorse.

[0104] In one embodiment, the complex is “autologous” to the vertebratesubject; that is, the complex is isolated from either from the infectedcells or the cancer cells or precancerous cells of the vertebratesubject (e.g., preferably prepared from infected tissues or tumorbiopsies of a vertebrate subject).

[0105] Alternatively, the complex is produced in vitro (e.g., wherein acomplex with an exogenous antigenic molecule is desired). Alternatively,GRP94 and/or the antigenic molecule can be isolated from a particularvertebrate subject, or from others, or by recombinant production methodsusing a cloned GRP94 originally derived from a particular vertebratesubject or from others. Exogenous antigens and fragments and derivatives(both peptide and non-peptide) thereof for use in complexing with GRP94(or in some instances HSP90), can be selected from among those known inthe art, as well as those readily identified by standard immunoassaysknow in the art by the ability to bind antibody or MHC molecules(antigenicity) or generate immune response (immunogenicity). Complexesof GRP94 and antigenic molecules can be isolated from cancer orprecancerous tissue of a subject, or from a cancer cell line, or can beproduced in vitro (as is necessary in the embodiment in which anexogenous antigen is used as the antigenic molecule).

[0106] D.1. Isolation of Antigenic/Immunogenic Components

[0107] A method for isolating or purifying an antigenic moleculeassociated with a complex comprising GRP94, or in some instances HSP90,is also provided in accordance with the present invention. In oneembodiment, the method comprises: contacting a sample comprising acomplex comprising an antigenic molecule and GRP94 with a binding agentthat preferentially binds GRP94, the binding agent immobilized to asolid phase support, to immobilize the complex to the solid phasesupport; collecting the remaining sample; eluting the complex from thesolid phase support to give purified complex in the eluate; andisolating the antigenic molecule from the eluate.

[0108] The binding agent preferably comprises an adenosine moiety orstructural mimetic thereof having any of a variety of substitutions atthe 2′, 3′, and 5′ positions, in the case of adenosine, as deemedappropriate by high resolution structural analyses of ligand-GRP94interactions. Optionally, 5′ position alkyl extensions can be included,preferably as a carboxamido linkage to the parent adenosine and, tofacilitate stable chemical linkage to a solid support for the purposesof affinity-based purification, terminating in any of a subset ofchemically reactive groups including, but not limited to vinyl,maleimide and/or succinimide esters, or substituents suitable forchemical coupling to solid phase supports, such as amino or sulphydrylgroups. More preferably, the binding agent is free of ATP or ADP. Arepresentative binding agent comprises a compound of formula (I) or acompound of formula (II). Another representative binding agent comprisesN-ethylcarboxamidoadenosine (NECA). Additional ligands can be identifiedthrough combinatorial chemistry of a parent precursor molecule bearing ahydrogen bond mimetic, preferably corresponding to the ribose ofadenosine, and a benzimidazole or structurally related scaffold,corresponding to the adenine base of adenosine.

[0109] Optionally, the complex bound to the immobilized binding agent iseluted by washing the solid phase support with a buffer comprising aphysiological salts solution containing appropriate concentrations ofthe parent ligand (i.e. the binding agent) to give complex in theeluate. Hence, a complex further comprising the binding agent or elutingligand is also provided in accordance with the present invention. Theeluting ligand will then be removed from the eluate solution by dialysisin buffers appropriate for GMP production including, but not limited to,physiological salts and volatile salts.

[0110] It has been found that antigenic peptides and/or components canbe eluted from GRP94-complexes under low pH conditions. Theseexperimental conditions can be used to isolate peptides and/or antigeniccomponents from cells which can contain potentially useful antigenicdeterminants. Once isolated, the amino acid sequence of each antigenicpeptide can be determined using conventional amino acid sequencingmethodologies. Such antigenic molecules can then be produced by chemicalsynthesis or recombinant methods; purified; and complexed to GRP94, oralternatively HSP90, in vitro. Additionally, antigenic peptide sequencescan be obtained by mass spectrometry using, but not limited to,electrospray and MALDI-TOF instrumentation, coupled with quadrapoledetection and CAD-based sequencing.

[0111] D.2. Elution of Peptides From GRP94-Peptide Complexes

[0112] Several methods can be used to elute a peptide from aGRP94-peptide complex or from a HSP90-peptide complex. The approachesinvolve incubating the complex in a low pH buffer and/or inguanidinium/HCl (3-6 M), 0.1-1% TFA or acetic acid. Briefly, the complexof interest is centrifuged through a CENTRICON®10 assembly (Amicon ofBeverly, Mass.) to remove any low molecular weight material looselyassociated with the complex. The large molecular weight fraction can beremoved and analyzed by SDS-PAGE while the low molecular weight materialis fractionated by capillary and/or nanoscale HPLC, with a flow rate of0.5 mL/min, with monitoring at 210/220 nm.

[0113] In the low pH protocol, acetic acid or trifluoroacetic acid (TFA)is added to the complex to give a final concentration of 10% (vol/vol)and the mixture incubated at room temperature or other suitabletemperature, for 10 minutes (Van Bleek et al. (1990) Nature 348:213-216;Li et al. (1993) EMBO J 12:3143-3151).

[0114] The resulting samples are centrifuged through a CENTRICON®10assembly as mentioned previously. The high and low molecular weightfractions are recovered. The remaining large molecular weight complexescan be reincubated with guanidinium or low pH to remove any remainingpeptides. The resulting lower molecular weight fractions are pooled,concentrated by evaporation and dissolved in 0.1% trifluoroacetic acid(TFA). The dissolved material is fractionated by microbore HPLC, with aflow rate of 0.5 ml/min. The elution of the peptides can be monitored byOD210/220nm and the fractions containing the peptides collected.

[0115] D.3. Sequencing and Synthesis of Peptides

[0116] The amino acid sequences of the eluted peptides can be determinedeither by manual or automated amino acid sequencing techniques wellknown in the art. Once the amino acid sequence of a potentiallyprotective peptide has been determined the peptide can be synthesized inany desired amount using conventional peptide synthesis or otherprotocols well known in the art.

[0117] A subject peptide can be synthesized by any of the techniquesthat are known to those skilled in the polypeptide art, includingrecombinant DNA techniques. Synthetic chemistry techniques, such as asolid-phase Merrifield-type synthesis, are preferred for reasons ofpurity, antigenic specificity, freedom from undesired side products,ease of production and the like. Many techniques for peptide synthesisare available and can be found in Steward et al. (1969) Solid PhasePeptide Synthesis, W. H. Freeman Co., San Francisco, Calif.; Bodanszky,et al. (1976) Peptide Synthesis, John Wiley & Sons, Second Edition;Meienhofer (1983) Hormonal Proteins and Peptides, Vol. 2, p. 46,Academic Press, New York, N.Y.; Merrifield (1969) Adv Enzymol32:221-296; Fields et al. (1990) Int J Peptide Protein Res 35:161-214;and U.S. Pat. No. 4,244,946 for solid phase peptide synthesis; andSchroder et al. (1965) The Peptides, Vol. 1, Academic Press, New York,N.Y. for classical solution synthesis, each of which is incorporatedherein by reference. Appropriate protective groups usable in suchsynthesis are described in the above texts and in McOmie (1973)Protective Groups in Organic Chemistry, Plenum Press, New York, N.Y.,which is incorporated herein by reference.

[0118] In general, the solid-phase synthesis methods contemplatedcomprise the sequential addition of one or more amino acid residues orsuitably protected amino acid residues to a growing peptide chain.Normally, either the amino or carboxyl group of the first amino acidresidue is protected by a suitable, selectively removable protectinggroup. A different, selectively removable protecting group is utilizedfor amino acids containing a reactive side group such as lysine.

[0119] Using a solid phase synthesis as exemplary, the protected orderivatized amino acid is attached to an inert solid support through itsunprotected carboxyl or amino group. The protecting group of the aminoor carboxyl group is then selectively removed and the next amino acid inthe sequence having the complimentary (amino or carboxyl) group suitablyprotected is admixed and reacted under conditions suitable for formingthe amide linkage with the residue already attached to the solidsupport. The protecting group of the amino or carboxyl group is thenremoved from this newly added amino acid residue, and the next aminoacid (suitably protected) is then added, and so forth. After all thedesired amino acids have been linked in the proper sequence, anyremaining terminal and side group protecting groups (and solid support)are removed sequentially or concurrently, to afford the final linearpolypeptide.

[0120] The resultant linear polypeptides prepared for example asdescribed above can be reacted to form their corresponding cyclicpeptides. An exemplary method for cyclizing peptides is described byZimmer et al. (1993) Peptides, pp. 393-394, ESCOM Science Publishers, B.V. Typically, tertbutoxycarbonyl protected peptide methyl ester isdissolved in methanol and sodium hydroxide solution are added and theadmixture is reacted at 20° C. to hydrolytically remove the methyl esterprotecting group. After evaporating the solvent, the tertbutoxycarbonylprotected peptide is extracted with ethyl acetate from acidified aqueoussolvent. The tertbutoxycarbonyl protecting group is then removed undermildly acidic conditions in dioxane cosolvent. The unprotected linearpeptide with free amino and carboxy termini so obtained is converted toits corresponding cyclic peptide by reacting a dilute solution of thelinear peptide, in a mixture of dichloromethane and dimethylformamide,with dicyclohexylcarbodiimide in the presence of 1-hydroxybenzotriazoleand N-methylmorpholine. The resultant cyclic peptide is then purified bychromatography.

[0121] Purification of the resulting peptides is accomplished usingconventional procedures, such as preparative HPLC using gel permeation,partition and/or ion exchange chromatography. The choice of appropriatematrices and buffers are well known in the art and so are not describedin detail herein.

[0122] D.4. Detection Methods

[0123] A method for detecting a complex comprising GRP94, or in someinstances HSP90, in a sample suspected of containing such a complex isalso provided in accordance with the present invention. In oneembodiment, the method comprises: contacting the sample with a bindingsubstance that preferentially binds GRP94 under conditions favorable tobinding a complex comprising GRP94 to the binding substance to form asecond complex there between; and detecting the second complex via alabel conjugated to the binding substance or via a labeled reagent thatspecifically binds to the second complex subsequent to its formation.

[0124] The binding substance preferably comprises an adenosine moiety orstructural mimetic thereof having any of a variety of substitutions atthe 2′, 3′, and 5′ positions, in the case of adenosine, as deemedappropriate by high resolution structural analyses of ligand-GRP94interactions. Optionally, 5′ position alkyl extensions can be included,preferably as a carboxamido linkage to the parent adenosine and, tofacilitate stable chemical linkage to a solid support for the purposesof affinity-based purification, terminating in any of a subset ofchemically reactive groups including, but not limited to vinyl,maleimide and/or succinimide esters, or substituents suitable forchemical coupling to solid phase supports, such as amino or sulphydrylgroups. More preferably, the binding substance is free of ATP or ADP. Arepresentative binding substance comprises a compound of formula (I) ora compound of formula (II). Another representative binding substancecomprises N-ethylcarboxamidoadenosine (NECA). Additional ligands can beidentified through combinatorial chemistry of a parent precursormolecule bearing a hydrogen bond mimetic, preferably corresponding tothe ribose of adenosine, and a benzimidazole or structurally relatedscaffold, corresponding to the adenine base of adenosine.

[0125] Optionally, the complex bound to the immobilized binding agent iseluted by washing the solid phase support with a buffer comprising aphysiological salts solution containing appropriate concentrations ofthe parent ligand (i.e. the binding substance or agent) to give complexin the eluate. Hence, a complex further comprising the binding agent oreluting ligand is also provided in accordance with the presentinvention. The eluting ligand will then be removed from the eluatesolution by dialysis in buffers appropriate for GMP productionincluding, but not limited to, physiological salts and volatile salts.

[0126] The binding substance can be conjugated with a detectable labeland in this case, the detecting step comprises: separating the complexfrom unbound labeled binding substance; and detecting the detectablelabel which is present in the complex or which is unbound.

[0127] D.5. Kits for Purification or Detection

[0128] In another aspect, the present invention pertains to a kit forisolating or purifying a peptide complex, preferably a GRP94 complex,and an antigenic molecule. In one embodiment, the kit comprises abinding agent that preferentially binds GRP94, the binding agentcontained in a first container. The binding agent preferably comprisesan adenosine moiety or structural mimetic thereof having any of avariety of substitutions at the 2′, 3′, and 5′ positions, in the case ofadenosine, as deemed appropriate by high resolution structural analysesof ligand-GRP94 interactions. Optionally, 5′ position alkyl extensionscan be included, preferably as a carboxamido linkage to the parentadenosine and, to facilitate stable chemical linkage to a solid supportfor the purposes of affinity-based purification, terminating in any of asubset of chemically reactive groups including, but not limited tovinyl, maleimide and/or succinimide esters, or substituents suitable forchemical coupling to solid phase supports, such as amino or sulphydrylgroups. More preferably, the binding agent is free of ATP or ADP.

[0129] A representative binding agent comprises a compound of formula(I) or a compound of formula (II). Another representative binding agentcomprises N-ethylcarboxamidoadenosine (NECA). Additional ligands can beidentified through combinatorial chemistry of a parent precursormolecule bearing a hydrogen bond mimetic, preferably corresponding tothe ribose of adenosine, and a benzimidazole or structurally relatedscaffold, corresponding to the adenine base of adenosine. Optionally,the binding agent can be immobilized to a solid phase support, or thekit can also comprise a solid phase support contained in a secondcontainer.

[0130] The kit can further comprise an elution buffer for use in elutinga complex from the binding agent, the elution buffer contained in athird container. Optionally, the elution buffer comprises aphysiological salts solution containing appropriate concentrations ofthe parent ligand to give complex in the eluate. The kit can furthercomprise dialysis buffers appropriate for GMP production including, butnot limited to, physiological salts and volatile salts. The kit can alsofurther comprise an elution buffer for use in eluting an antigenicmolecule from a complex, the elution buffer contained in a fourthcontainer. Suitable elution buffers are disclosed herein above.

[0131] In the case of a kit used for detecting a complex comprisingGRP94, or alternatively a complex comprising the kit can furthercomprise a reagent or indicator that comprises a detectable label, theindicator containing in a fifth container. Alternatively, the bindingagent can comprise a detectable label or indicator. The indicator cancomprise a radioactive label or an enzyme, or other indicator asdisclosed herein.

[0132] D.6. Determination of Immunogenicity of GRP94-Peptide Complexes

[0133] Purified GRP94-antigenic molecule complexes can be assayed forimmunogenicity using the mixed lymphocyte tumor culture assay (MLTC)well known in the art. By way of example but not limitation, thefollowing procedure can be used. Briefly, mice are injectedsubcutaneously with the candidate GRP94-antigenic molecule complexes.Other mice are injected with either other GRP94-antigenic moleculecomplexes or whole infected cells which act as positive controls for theassay. The mice are injected twice, 7-10 days apart. Ten days after thelast immunization, the spleens are removed and the lymphocytes released.The released lymphocytes can be re-stimulated subsequently in vitro bythe addition of dead cells that expressed the complex of interest.

[0134] For example, 8×10⁶ immune spleen cells can be stimulated with4×10⁴ mitomycin C treated or γ-irradiated (5-10,000 rads) infected cells(or cells transfected with an appropriate gene, as the case can be) in 3ml RPMI medium containing 10% fetal calf serum. In certain cases 33%secondary mixed lymphocyte culture supernatant can be included in theculture medium as a source of T cell growth factors, such as isdescribed by Glasebrook et al. (1980) J Exp Med 151:876. To test theprimary cytotoxic T cell response after immunization, spleen cells canbe cultured without stimulation. In some experiments spleen cells of theimmunized mice can also be re-stimulated with antigenically distinctcells, to determine the specificity of the cytotoxic T cell response.

[0135] Six days later the cultures are tested for cytotoxicity in a 4hour ⁵¹Cr-release assay as is described by Palladino et al. (1987)Cancer Res 47:5074-5079 and Blachere et al. (1993) J Immunotherapy14:352-356. In this assay, the mixed lymphocyte culture is added to atarget cell suspension to give different effector:target (E:T) ratios(usually 1:1 to 40:1). The target cells are prelabeled by incubating1×10⁶ target cells in culture medium containing 200 mCi ⁵¹Cr/ml for onehour at 37° C. The cells are washed three times following labeling. Eachassay point (E:T ratio) is performed in triplicate and the appropriatecontrols incorporated to measure spontaneous ⁵¹Cr release (nolymphocytes added to assay) and 100% release (cells lysed withdetergent). After incubating the cell mixtures for 4 hours, the cellsare pelleted by centrifugation at 200 g for 5 minutes. The amount of⁵¹Cr released into the supernatant is measured by a gamma counter. Thepercent cytotoxicity is measured as cpm in the test sample minusspontaneously released cpm divided by the total detergent released cpmminus spontaneously released cpm.

[0136] In order to block the MHC class I cascade a concentratedhybridoma supernatant derived from K-44 hybridoma cells (an anti-MHCclass I hybridoma) is added to the test samples to a final concentrationof 12.5%.

[0137] E. Screening Methods

[0138] Disclosed herein is the molecular basis, as well as a highthroughput screen, for chemical compounds that elicit or inhibitconformational changes in the molecular chaperone GRP94, or in someinstances HSP90, thereby regulating the chaperone and peptide bindingactivities of these proteins.

[0139] Also disclosed herein are several new and unique aspects of theregulation of GRP94 structure and function that can be readily exploitedfor purposes of identifying agonists and antagonists (“modulators”) ofGRP94 function. GRP94 expression is upregulated by cellular stressessuch as nutrient deprivation, oxidative stress, heavy metal posioning,hypoxia/anoxia, and other conditions related to ischemia. However, untilthe disclosure of the present invention, the molecular mechanismunderlying this activity remained unknown. Thus, disclosed herein is afunctional correlation to heat shock in the observation that heat shockstimulates the peptide binding and chaperone activity of GRP94. The heatshock response of GRP94, which is responsible for its increased peptidebinding and chaperone activity, is a result of a change in theconformational state of the protein from a closed form to an open,active form.

[0140] The heat shock induced conformational change can be blocked bythe antitumor drugs geldanamycin and radicicol, thus providing amechanism of their antitumor activity, namely that geldanamycin andradicicol block GRP94 conformational transitions, and hence chaperoneactivity. The functional consequence of such inhibition is thatoncogenic signaling proteins, such as growth factor receptor kinases arenot processed properly and thus, the cell does not receive theproliferative signals necessary for transformation. Thus, a chemicalcompound that modulates the conformation of GRP94 can be used to treat adisease state, such as cancer, wherein a therapeutic benefit can beprovided by inhibiting or blocking the egress of proteins (e.g., growthfactors) from the endoplasmic reticulum.

[0141] The present invention provides the theoretical and structuralbasis for the identification of low molecular weight molecules that bindto a recently crystallized conserved N-terminal domain of HSP90, whichpreviously was identified as the binding site for the anti-tumor druggeldanamycin, and elicit a conformation change that yields a dramaticand substantial increase in (poly)peptide binding activity of GRP94, andin some cases, HSP90. In an alternative embodiment, the identifiedmolecules inhibit conformational activation of GRP94, and in some casesHSP90, similar to the observed modulation of GRP94 and HSP90 bygeldanamycin and/or radicicol.

[0142] The present invention is markedly distinguished from currentperception in the art as to the mechanism of regulation of GRP94 andHSP90 function. In current views, the Hsp90 family of molecularchaperones are thought to be regulated by cycles of ATP binding andhydrolysis (Prodromou et al. (1997) Cell 90:65-75). This view of Hsp90function is based on the observations that the highly conservedN-terminal domain of the protein contains a binding site for ATP and ADPand that X-ray crystallographic structures of the domain in complex withATP and/or ADP can be obtained.

[0143] In accordance with the present invention, data are provideddemonstrating that the related and relevant domain of the HSP90 paralogGRP94 does not display a specific structural preference for ATP or ADP.In a series of function-directed studies, applicants have furtherdetermined that ATP, ADP, geldanamycin and radicicol block or inhibitthe ability of GRP94 to assume a conformation necessary for chaperoneactivity and/or peptide binding. Thus, ATP and ADP, rather than beingphysiological ligands agonising the activity of GRP94, act as inhibitoryagents for this chaperone.

[0144] The identified conformational change in GRP94 is a component ofthe regulatory cycle of GRP94, as demonstrated in the Examples whereinbis-ANS, which bears structural similarities to adenosine nucleotides,was demonstrated to elicit a tertiary conformational change in GRP94that was accompanied by an activation of molecular chaperone and peptidebinding activity.

[0145] In accordance with the present invention, also disclosed hereinare the primary structural determinants that define low molecular weightcompounds that bind to the conserved N-terminal domain of GRP94 andeither A) elicit a conformational change in GRP94 that is accompanied byan activation of either peptide binding and/or molecular chaperoneactivity, or B) block or inhibit the ability of GRP94 to access oracquire the described conformation. In the present invention, and aswould be apparent to one of ordinary skill in the art of the regulationof protein structure/function after reviewing the disclosure presentedherein, cells and tissues originating from higher eukaryotes contain anative ligand compound bearing structural similarities to adenosine, yetmay bear substituents at the 2′ and 5′ positions, but lack substituentsat the N6 adenine.

[0146] Thus, a native ligand, as well an embodiment of mimetic thereof,bears an adenosine moiety or moieties and the adenosine moiety(s)function in the binding of the ligand to the conserved N-terminal domainof GRP94 previously identified as an ATP/ADP binding pocket.Representative ligand compositions are disclosed herein above asformulas (I) and (II). Additional ligands can be identified throughcombinatorial chemistry of a parent precursor molecule bearing ahydrogen bond mimetic, preferably corresponding to the ribose ofadenosine, and a benzimidazole or structurally related scaffold,corresponding to the adenine base of adenosine.

[0147] The binding of a ligand elicits the conformational change that isaccompanied by an activation of chaperone and peptide binding activity.Furthermore, synthesis of the native ligand is likely stimulated byconditions that elicit a disruption in the efficiency of protein foldingand assembly in the ER. These conditions include, but are not limitedto, heat shock, oxidative stress, nutrient deprivation, disruptions inoligosaccharide synthesis and covalent assembly on to nascentglycoproteins, and the presence of excessive levels of heavy metals.

[0148] Coincident with the discovery of the functional role for GRP94structural transitions in determining the chaperone activity and themechanism of geldanamycin and radicicol action, a simple and rapidmethod for assaying the conformational state of GRP94 (or alternatively,HSP90) is disclosed herein. A preferred embodiment of this method isbased on the preferential binding of the small synthetic fluorescentprobe, bis-ANS, to the open, or active, conformation of GRP94. bis-ANSbinding yields a dramatic increase in probe fluorescence intensity.bis-ANS is identified herein as a highly sensitive indicator of the heatshock induced conformational change of GRP94. Furthermore, bis-ANSitself can elicit the conformational change in GRP94 necessary for theactivation of peptide binding and chaperone function. Thus, bis-ANS isboth an agonist for GRP94 activation as well as an indicator for therelative state of activation. bis-ANS induces these changes on a slowtime scale, thereby enabling it to be used both as an inducer for a heatshock-like conformational change as well as a probe for conformationalchanges induced by other compounds. Conversely, and as disclosed in theExamples, bis-ANS can be used to identify compounds that block the heatshock-induced conformational changes. Indeed, the screening system ofthe present invention showed that radicicol and geldanamycin, twoanti-tumor agents known to act through GRP94/HSP90, block the conversionof these proteins to the conformation necessary for function.

[0149] Another preferred embodiment of this method employs a relatedsynthetic fluorescent probe, 8-ANS. 8-ANS also displays preferentialbinding to the active conformation of GRP94. However, unlike bis-ANS,8-ANS functions solely as an indicator and lacks agonist activity. 8-ANSis also useful in screening assays for discovery of GRP94 modulators.

[0150] Therefore, in accordance with the present invention, a method ofscreening candidate compounds for an ability to modulate the biologicalactivity is provided. The screening methods are also used to identify anative or endogenous ligand or ligands for GRP94.

[0151] In one embodiment, a candidate substance is a substance whichpotentially can modulate the biological activity of GRP94 by binding orother intermolecular interaction with GRP94. By “modulate” is intendedan increase, decrease, or other alteration of any or all biologicalactivities or properties of GRP94. Thus, a native or endogenous ligandor ligands of GRP94 is also a “candidate substance”. A biological samplesuspected of containing a native or endogenous ligand or ligands is alsoa “candidate substance”. Small molecules and combinatorial libraries ofsmall molecules are also candidate “substances”. A candidate substanceidentified according to a screening assay described herein has theability to modulate GRP94 biological activity. Such a candidatesubstance has utility in the treatment of disorders and conditionswherein modulation of the biological activity of GRP94 is desirable, aswell as in the purification and screening methods disclosed herein.

[0152] The present invention thus pertains to the molecular basis for aswell as a high throughput screen for chemical compounds that elicit orinhibit conformational changes in the molecular chaperone GRP94, or insome instances HSP90, thereby regulating the chaperone and peptidebinding activities of these proteins.

[0153] E.1. General Screening Methods

[0154] A method of screening candidate substances for an ability tomodulate GRP94 and/or HSP90 biological activity is thus provided inaccordance with the present invention. In one embodiment, the methodcomprises (a) establishing a test sample comprising GRP94 and a ligandfor GRP94; (b) administering a candidate substance or a sample suspectedof containing a candidate substance to the test sample; and (c)measuring an effect on binding of GRP94 and the ligand for GRP94 in thetest sample to thereby determine the ability of the candidate substanceto modulate GRP94 biological activity.

[0155] The test sample can further comprise an indicator. The term“indicator” is meant to refer to a chemical species or compound that isreadily detectable using a standard detection technique, such as darkversus light detection, fluorescence or chemiluminescencespectrophotometry, scintillation spectroscopy, chromatography, liquidchromatography/mass spectroscopy (LC/MS), colorimetry, and the like.Representative indicator compounds thus include, but are not limited to,fluorogenic or fluorescent compounds, chemiluminescent compounds,calorimetric compounds, UV/VIS absorbing compounds, radionucleotides andcombinations thereof. In a preferred embodiment, the ligand furthercomprises an indicator. In a more preferred embodiment, theligand/indicator comprises 1,8-anilinonapthalenesulfonate (8-ANS).

[0156] The ability of the candidate substance to modulate GRP94 and/orHSP90 biological activity can determined in any suitable manner. Forexample, the ability of the candidate substance to modulate GRP94 and/orHSP90 biological activity can determined by: (i) detecting a signalproduced by the indicator upon an effect of the candidate substance onbinding of GRP94 and/or HSP90 and the ligand for GRP94 and/or HSP90; and(ii) identifying the candidate substance as a modulator of GRP94 and/orHSP90 biological activity based upon an amount of signal produced ascompared to a control sample.

[0157] In a preferred embodiment, a simple and effective fluorescencebased screening methodology is provided to identify inhibitors andactivators of the conformational transitions of GRP94 which areresponsible for its activity. The method is readily amenable to bothrobotic and very high throughput systems.

[0158] Thus, in one embodiment, a screening method of the presentinvention pertains to a method for a identifying a candidate substanceas an activator of the biological activity of an Hsp90 protein. In apreferred embodiment, the Hsp90 protein is GRP94 or HSP90. The methodcomprises establishing a test sample comprising an Hsp90 protein and acandidate substance; administering 8-ANS to the test sample; anddetecting a fluorescence signal produced by the 8-ANS; and identifyingthe candidate substance as an activator of the biological activity ofthe Hsp90 protein based upon an amount of fluorescence signal producedby the 8-ANS as compared to a control sample.

[0159] The method can further comprise incubating the Hsp90 protein withthe candidate substance at 37° C for about one hour prior to adding the8-ANS. Optionally, the 8-ANS can be added in an approximately equimolaramount to the Hsp90 protein. Additionally, the candidate substance isidentified as an activator of the biological activity of an Hsp90protein by detection of an increased 8-ANS fluorescence signal ascompared to a control sample.

[0160] In another embodiment, a screening method of the presentinvention pertains to a method for a identifying a candidate substanceas an inhibitor of the biological activity of a Hsp90 protein. Themethod comprises establishing a test sample comprising an Hsp90 proteinand a candidate substance; heat-shocking the test sample to induce aconformational change to the Hsp90 protein; administering 8-ANS to thetest sample; detecting a fluorescence signal produced by the 8-ANS; andidentifying the candidate substance as an inhibitor of the biologicalactivity of an Hsp90 protein based upon an amount of fluorescence signalproduced by the 8-ANS as compared to a control sample. In a preferredembodiment, the Hsp90 protein is GRP94 or HSP90.

[0161] Optionally, the method can further comprise incubating the testsample at 37° C. for about one hour prior to heat-shocking the testsample. The heat-shocking can be carried out at 50° C. for about 15minutes. Preferably, the 8-ANS is added in an approximately equimolaramount to the Hsp90 protein. The candidate substance can also beidentified as an inhibitor of the biological activity of an Hsp90protein by detection of a decreased 8-ANS fluorescence signal ascompared to a control sample.

[0162] E.2. Cell Based Screening Assays

[0163] A screening assay of the present invention may also involvedetermining the ability of a candidate substance to modulate, i.e.inhibit or promote the biological activity of an Hsp90 protein such asGRP94 and preferably, to thereby modulate the biological activity of anHsp90 protein such as GRP94 in target cells. Target cells can be eithernaturally occurring cells known to contain a polypeptide of the presentinvention or transformed cells produced in accordance with a process oftransformation set forth herein above. The test samples can furthercomprise a cell or cell line that expresses an Hsp90 polypeptide; thepresent invention also contemplates a recombinant cell line suitable foruse in the exemplary method. Such cell lines may be mammalian, or human,or they may from another organism, including but not limited to yeast.

[0164] Representative assays including genetic screening assays andmolecular biology screens such as a yeast two-hybrid screen that willeffectively identify Hsp90-interacting genes important for Hsp90 orother Hsp90-mediated cellular process, including a native Hsp90 ligandor ligands. One version of the yeast two-hybrid system has beendescribed (Chien et al. (1991) Proc Natl Acad Sci USA 88:9578-9582) andis commercially available from Clontech (Palo Alto, Calif.). Thus, inaccordance with one embodiment of a screening assay of the presentinvention, the candidate substance is further characterized as acandidate polypeptide, and the screening method can further comprise thestep of purifying and isolating a nucleic acid molecule encoding thecandidate polypeptide.

[0165] Thus, enzymes in the cells of higher eukaryotes that mediate thesteady state and stress-elicited production of a GRP94 and/or HSP90ligand can also be modulated in accordance with the present invention.Such catabolic enzymes also represent appropriate and rational targetsfor the design of compounds that elicit an increase in the steady statelevels of a native Hsp90 ligand (e.g., a native GRP94 or HSP90 ligand)and thereby lead to the elicitation of the structural and functionalactivation of chaperone and peptide binding activity of an Hsp90protein, preferably GRP94, disclosed herein.

[0166] A screening assay can provide a cell under conditions suitablefor testing the modulation of biological activity of an Hsp90 proteinsuch as GRP94. These conditions include but are not limited to pH,temperature, tonicity, the presence of relevant metabolic factors (e.g.,metal ions such as for example Ca⁺⁺, growth factor, interleukins, orcolony stimulating factors), and relevant modifications to thepolypeptide such as glycosylation or prenylation. A polypeptide of thepresent invention can be expressed and utilized in a prokaryotic oreukaryotic cell. The host cell can also be fractionated intosub-cellular fractions where the receptor can be found. For example,cells expressing the polypeptide can be fractionated into the nuclei,the endoplasmic reticulum, vesicles, or the membrane surfaces of thecell. U.S. Pat. Nos. 5,837,479; 5,645,999; 5,786,152; 5,739,278; and5,352,660 also describe exemplary screening assays, and the entirecontents of each are herein incorporated by reference.

[0167] E.3. High Throughput Screening

[0168] In another embodiment of the screening method of the presentinvention, an Hsp90 polypeptide (e.g., human GRP94) or active fragmentor oligopeptide thereof, can be used for screening libraries ofcompounds in any of a variety of high throughput drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, between theHsp90 polypeptide, preferably a GRP94 polypeptide, and the candidatesubstance being tested, can be measured as described herein.

[0169] E.4. Rational Drug Design

[0170] A method of identifying modulators of an Hsp90 protein byrational drug design is also provided in accordance with the presentinvention. The method comprises designing a potential modulator for anHsp90 protein that will form non-covalent bonds with amino acids in thesubstrate binding site based upon the structure of an Hsp90 proteinpreferably GRP94; synthesizing the modulator; and determining whetherthe potential modulator modulates the activity of an Hsp90 protein.Modulators may be synthesized using techniques known in the art. Thedetermination of whether the modulator modulates the biological activityof an Hsp90 protein is made in accordance with the screening methodsdisclosed herein, or by other screening methods known in the art. Thisis the method of “rational” drug design.

[0171] Additional representative rational drug design techniques aredescribed in U.S. Pat. Nos. 5,834,228 and 5,872,011, the entire contentsof which are herein incorporated by reference.

[0172] Thus, a method of identifying modulators of an Hsp90 protein byrational drug design is provided in accordance with the presentinvention. The method comprises designing a potential modulator for anHsp90 protein that will form non-covalent bonds with amino acids in theHsp90 protein substrate binding site based upon a crystal structure ofan Hsp90 protein; synthesizing the modulator; and determining whetherthe potential modulator modulates the activity of an Hsp90 protein.Modulators are synthesized using techniques disclosed herein and as areknown in the art. The determination of whether the modulator modulatesthe biological activity of an Hsp90 protein is made in accordance withthe screening methods disclosed herein above. In a preferred embodiment,the Hsp90 protein is GRP94.

[0173] F. Modulation of Hsp90 Biological Activity

[0174] Because Hsp90 proteins are found in essentially every cell of thehuman body and are involved in the processing of many different cellularproteins as well as the presentation of tumor and foreign antigens tothe immune system, compounds identified through the screening method ofthe present invention and disclosed herein (referred to as “ligandcompositions” or “modulators”) have wide ranging value as therapeuticsand in vaccine development. Representative ligand compositions ormodulators are described herein above as formula (I). Modulators that donot structurally resemble adenosine are also provided, and include thosedesigned and/or identified by the rational drug design and combinatorialscreening methods disclosed hereinabove.

[0175] In a preferred embodiment, the Hsp90 modulator elicits aconformational change in an Hsp90 protein. Even more preferably, theHsp90 protein activity modulator is identified according to a screeningassay described herein. A modulator can modulate the biological activityof an Hsp90 protein such as GRP94. Relevant to the antigen-presentingactivity of GRP94 and HSP90, activators thereof can be applied in vitroto assist in peptide loading onto these proteins for the production ofvaccines directed against the tissues or invasive organisms possessingthose specific peptide epitopes. Activators of GRP94/HSP90 biologicalactivity can be applied to tumor cells excised from cancer patients toincrease the antigenicity of the tumor cells prior to lethalinactivation of the cells and their re-injection into the body asimmunostimulatory agents. Activators of GRP94/HSP90 biological activitycan be administered directly into the body of a vertebrate forincreasing the antigenicity of tumors in situ. Activators of GRP94/HSP90biological activity can also have antibiotic action against bacteria,viruses, or internal parasites by increasing the antigenicity of thebacteria, virus, or parasites and recognition of same by the adaptiveimmune system. Activators of GRP94/HSP90 biological activity can be usedin further screens to identify peptides from combinatorial librarieswhich represent specific anti-tumor, anti-viral, or anti-bacterialepitopes. Relevant to the chaperone activity of GRP94 and HSP90,activators thereof can also ameliorate or prevent cellular damageresulting from ischemic conditions.

[0176] Inhibitors of GRP94/HSP90 function can possess anti-tumoractivity. Inhibitors of GRP94/HSP90 function can also interfere with theprocessing of viral or bacterial proteins in infectious states and slowthe progress of these infections. Inhibitors of GRP94/HSP90 function canalso be administered to a vertebrate subject to decrease theantigenicity of tissues to alleviate transplanted tissue rejection oreven slow the progression of autoimmune diseases such as rheumatoidarthritis and systemic lupus erythramatosis. Inhibitors of GRP94activity can also be used for treatment of diseases, such as cancer, byinhibiting or blocking the egress of proteins (e.g., growth factors)from the endoplasmic reticulum.

[0177] A biological activity of a Hsp90 protein such as GRP94 that ismodulated in accordance with the present invention can include, but isnot limited to, loading activity in the formation of a complex withantigenic molecules, eliciting an immune response in a subject; treatingor preventing a type of cancer in a subject; treating or preventing aninfectious disease in a subject; sensitizing antigen presenting cells(APC), particularly with respect to a type of cancer or an infectiousdisease; and enhancing protein transport along the endoplasmicreticulum.

[0178] Another modulatable biological activity of a Hsp90 proteincomprises preventing or ameliorating cellular damage arising fromconditions of ischemia/reperfusion including but not limited to cardiacarrest, asystole and sustained ventricular arrythmias, cardiac surgery,cardiopulmonary bypass surgery, organ transplantation, spinal cordinjury, head trauma, stroke, thromboembolic stroke, hemorrhagic stroke,cerebral vasospasm, hypotension, hypoglycemia, status epilepticus, anepileptic seizure, anxiety, schizophrenia, a neurodegenerative disorder,Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis(ALS), or neonatal stress. In this case, a ligand can modulate anendogenous Hsp90 protein by promoting conformational activation of theHsp90 protein. Preferably, the ligand was identified according to ascreening or rational drug design method disclosed herein and isrelevant for the modulation of GRP94 or HSP90.

[0179] F.1. In vitro Production of GRP94-Antigenic Molecule Complexes

[0180] In accordance with the present invention, complexes of an Hsp90protein, such as GRP94, to antigenic molecules are produced in vitrousing an Hsp90 protein activity modulator. As will be appreciated bythose skilled in the art, the peptides either isolated by proceduresdisclosed herein, chemically synthesized or recombinantly produced, canbe reconstituted with a variety of naturally purified or recombinantHsp90 proteins in vitro to generate, for example, immunogenicnon-covalent GRP94-antigenic molecule complexes. Alternatively,exogenous antigens or antigenic/immunogenic fragments or derivativesthereof can be non-covalently complexed to an Hsp90 protein for use inthe immunotherapeutic or prophylactic vaccines of the invention. Thecomplexes can then be purified using any suitable method, and arepreferably purified via the affinity purification methods of the presentinvention disclosed herein above.

[0181] In a representative approach, antigenic molecules (1 μg) andGRP94 (9 μg) are admixed to give an approximately 5 antigenic molecule:1 GRP94 molar ratio. Then, the mixture is incubated for 15 minutes to 3hours at 4° C. to 45° C. with bis-ANS in a quantity equimolar to GRP94in a suitable binding buffer such as one containing 20 mM sodiumphosphate, pH 7.2, 350 mM NaCl, 3 mM MgCl₂ and 1 mM phenyl methylsulfonyl fluoride (PMSF). The preparations are centrifuged throughCENTRICON®10 assembly (Amicon of Beverly, Mass.) to remove any unboundpeptide. The association of the peptides with GRP94 can be assayed bySDS-PAGE. Additional representative approaches are disclosed in theExamples.

[0182] Following complexing, the immunogenic GRP94-antigenic moleculecomplexes can optionally be assayed in vitro using, for example, themixed lymphocyte tumor cell assay (MLTC) described herein. Onceimmunogenic complexes have been isolated they can be optionallycharacterized further in animal models using the preferredadministration protocols and excipients discussed herein.

[0183] F.1 .1. Exogenous Antigenic Molecules

[0184] Antigens or antigenic portions thereof can be selected for use asantigenic molecules, for complexing to an Hsp90 protein, such as GRP94,from among those known in the art or determined by immunoassay to beable to bind to antibody or MHC molecules (antigenicity) or generateimmune response (immunogenicity). To determine immunogenicity orantigenicity by detecting binding to antibody, various immunoassaysknown in the art can be used, including but not limited to competitiveand non-competitive assay systems using techniques such asradioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitinreactions, immunodiffusion assays, in vivo immunoassays (using colloidalgold, enzyme or radioisotope labels, for example), western blots,immunoprecipitation reactions, agglutination assays (e.g., gelagglutination assays, hemagglutination assays), complement fixationassays, immunofluorescence assays, protein A assays, andimmuno-electrophoresis assays, etc.

[0185] In one embodiment, antibody binding is detected by detecting alabel on the primary antibody. In another embodiment, the primaryantibody is detected by detecting binding of a secondary antibody orreagent to the primary antibody. In a further embodiment, the secondaryantibody is labeled. Many methods and techniques are known in the artfor detecting binding in an immunoassay and can be used. In oneembodiment for detecting immunogenicity, T cell-mediated responses canbe assayed by standard methods, e.g., in vitro cytotoxicity assays or invivo delayed-type hypersensitivity assays.

[0186] Potentially useful antigens or derivatives thereof for use asantigenic molecules can also be identified by various criteria, such asthe antigen's involvement in neutralization of a pathogen's infectivity(wherein it is desired to treat or prevent infection by such a pathogen)(Norrby (1985) “Summary” in Vaccines 85, Lerner et al. (eds.), pp.388-389, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.), type orgroup specificity, recognition by subjects antisera or immune cells,and/or the demonstration of protective effects of antisera or immunecells specific for the antigen. In addition, where it is desired totreat or prevent a disease caused by a pathogen, the antigen's encodedepitope should preferably display a small or no degree of antigenicvariation in time or amongst different isolates of the same pathogen.

[0187] Preferably, where it is desired to treat or prevent cancer, knowntumor-specific antigens or fragments or derivatives thereof are used.For example, such tumor specific or tumor-associated antigens includebut are not limited to KS ¼ pan-carcinoma antigen (Perez & Walker (1990)J Immunol 142:3662-3667; Bumal (1988) Hybridoma 7(4):407-415); ovariancarcinoma antigen (CA125) (Yu et al. (1991) Cancer Res 51(2):468-475);prostatic acid phosphate (Tailer et al. (1990) Nuc Acids Res18(16):4928); prostate specific antigen (Henttu & Vihko (1989) BiochemBiophys Res Comm 160(2):903-910; Israeli et al. (1993) Cancer Res53:227-230); melanoma-associated antigen p97 (Estin et al. (1989) J NatlCancer Inst 81(6):445-446); melanoma antigen gp75 (Vijayasardahl et al.(1990) J Exp Med 171(4):1375-1380); high molecular weight melanomaantigen (Natali et al. (1987) Cancer59:55-63) and prostate specificmembrane antigen. In a specific embodiment, an antigen or fragment orderivative thereof specific to a certain tumor is selected forcomplexing to an Hsp90 protein, such as GRP94, and subsequentadministration to a subject having that tumor.

[0188] Preferably, where it is desired to treat or prevent viraldiseases, molecules comprising epitopes of known viruses are used. Forexample, such antigenic epitopes can be prepared from viruses including,but not limited to, hepatitis type A hepatitis type B, hepatitis type C,influenza, varicella, adenovirus, herpes simplex type I (HSV-I), herpessimplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus,respiratory syncytial virus (RSV), papilloma virus, papova virus,cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus,mumps virus, measles virus, rubella virus, polio virus, humanimmunodeficiency virus type I (HIV-I), and human immunodeficiency virustype II (HIV-II). Preferably, where it is desired to treat or preventbacterial infections, molecules comprising epitopes of known bacteriaare used. For example, such antigenic epitopes can be prepared frombacteria including, but not limited to, Mycobacteria, Mycoplasma,Neisseria, and Legionella.

[0189] Preferably, where it is desired to treat or prevent protozoalinfectious, molecules comprising epitopes of known protozoa are used.For example, such antigenic epitopes can be prepared from protozoaincluding, but not limited to, Leishmania, Kokzidioa, and Trypanosoma.Preferably, where it is desired to treat or prevent parasiticinfectious, molecules comprising epitopes of known parasites are used.For example, such antigenic epitopes can be from parasites including,but not limited to, Chlamydia and Rickettsia.

[0190] F.1.2. Peptides from MHC Complexes

[0191] Candidate immunogenic or antigenic peptides can be isolated fromeither endogenous Hsp90-peptide complexes as described above or fromendogenous MHC-peptide complexes for use subsequently as antigenicmolecules, by complexing in vitro to an Hsp90 protein, such as GRP94.The isolation of potentially immunogenic peptides from MHC molecules iswell known in the art and so is not described in detail herein. See Falket al. (1990) Nature 348:248-251; Rotzsche et al. (1990) Nature348:252-254; Elliott et al. (1990) Nature 348:191-197; Falk et al.(1991) Nature 351:290-296; Demotz et al. (1989) Nature 343:682-684;Rotzsche et al. (1990) Science 249:283-287, the disclosures of which areincorporated herein by reference. Briefly, MHC-peptide complexes can beisolated by a conventional immuno-affinity procedure. The peptides canthen be eluted from the MHC-peptide complex by incubating the complexesin the presence of about 0.1% TFA in acetonitrile. The eluted peptidescan be fractionated and purified by HPLC as described herein.

[0192] F.2. Therapeutic Methods for Modulating Hsp90 Biological Activity

[0193] A therapeutic method according to the present invention comprisesadministering to a subject in need thereof a substance that modulates,i.e., inhibits or promotes, biological activity of an Hsp90 protein,such as GRP94. Representative substances, also referred to as “ligandcompositions” or “modulators” are disclosed herein (e.g., compounds offormula (I)) and can also be identified according to any of thescreening assays set forth herein. The method comprises treating asubject suffering from a disorder wherein modulation of the biologicalactivity of an Hsp90 protein is desirable by administering to thesubject an effective amount of an Hsp90 modulator. Preferably, the Hsp90protein is GRP94. More preferably, the modulator elicits aconformational change in an Hsp90 protein. Even more preferably, themodulator is identified according to a screening assay described herein.

[0194] By the term “modulating”, it is meant that the substance caneither promote or inhibit the biological activity of an Hsp90 protein,depending on the disorder to be treated, and can affect one or severalof the Hsp90 proteins, including GRP94. Administration can providetreatment of disorders which can be exacerbated by GRP94/HSP90-mediatedmechanisms, including but not limited to, cancer, infectious diseases,and ischemic conditions.

[0195] The subject treated in the present invention in its manyembodiments is desirably a human subject, although it is to beunderstood that the principles of the invention indicate that theinvention is effective with respect to invertebrate and to allvertebrate species, including mammals, which are intended to be includedin the term “subject”. This is particularly the case in view of thephylogenetically ubiquitous nature of Hsp90 proteins. Moreover, a mammalis understood to include any mammalian species in which treatment orprevention of cancer or infectious diseases is desirable, particularlyagricultural and domestic mammalian species.

[0196] The methods of the present invention are particularly useful inthe treatment of warm-blooded vertebrates. Therefore, the inventionconcerns mammals and birds.

[0197] More particularly, contemplated is the treatment of mammals suchas humans, as well as those mammals of importance due to beingendangered (such as Siberian tigers), of economical importance (animalsraised on farms for consumption by humans) and/or social importance(animals kept as pets or in zoos) to humans, for instance, carnivoresother than humans (such as cats and dogs), swine (pigs, hogs, and wildboars), ruminants (such as cattle, oxen, sheep, giraffes, deer, goats,bison, and camels), and horses. Also contemplated is the treatment ofbirds, including the treatment of those kinds of birds that areendangered, kept in zoos, as well as fowl, and more particularlydomesticated fowl, i.e., poultry, such as turkeys, chickens, ducks,geese, guinea fowl, and the like, as they are also of economicalimportance to humans. Thus, contemplated is the treatment of livestock,including, but not limited to, domesticated swine (pigs and hogs),ruminants, horses, poultry, and the like.

[0198] In one embodiment, a ligand composition or modulator isadministered in conjunction with a complex comprising an Hsp90 protein(preferably GRP94 or HSP90) and an antigenic molecule. Preferably, thecomplex is “autologous” to the subject; that is, the complex is isolatedfrom either from the infected cells or the cancer cells or precancerouscells of the subject (e.g., preferably prepared from infected tissues ortumor biopsies of a subject). More preferably, the complex is purifiedin accordance with a purification method of the present inventiondisclosed herein above.

[0199] Alternatively, the complex is produced in vitro (e.g., wherein acomplex with an exogenous antigenic molecule is desired). Alternatively,the Hsp90 protein (preferably GRP94 or HSP90) and/or the antigenicmolecule can be isolated from a particular subject or from others or byrecombinant production methods using a cloned Hsp90 protein (preferablyGRP94 or HSP90) originally derived from a particular subject or fromothers. Exogenous antigens and fragments and derivatives (both peptideand non-peptide) thereof for use in complexing with an Hsp90 protein,can be selected from among those known in the art, as well as thosereadily identified by standard immunoassays know in the art by theability to bind antibody or MHC molecules (antigenicity) or generateimmune response (immunogenicity). Complexes of an Hsp90 protein(preferably GRP94 or HSP90) and antigenic molecules can be isolated fromcancer or precancerous tissue of a subject, or from a cancer cell line,or can be produced in vitro (as is necessary in the embodiment in whichan exogenous antigen is used as the antigenic molecule). Preferably, thecomplex is purified in accordance with a purification method of thepresent invention disclosed herein above.

[0200] The invention also provides a method for measuring tumorrejection in vivo in a subject, preferably a human subject, comprisingmeasuring the generation by the subject of MHC Class I-restricted CD8⁺cytotoxic T lymphocytes specific to the tumor after administering acomplex comprising GRP94 and antigenic molecules specific to the tumorin conjunction with an GRP94 biological activity modulator. Preferably,GRP94 comprises human GRP94. The immunogenic GRP94-peptide complexes ofthe invention can include any complex containing a GRP94 and a peptidethat is capable of inducing an immune response in a subject. Thepeptides are preferably non-covalently associated with the GRP94.

[0201] Although the Hsp90 protein can be allogenic to the subject (e.g.,isolated from cancerous tissue from a second vertebrate subject that isthe same type as a cancerous tissue present in a first vertebratesubject to be treated), in a preferred embodiment, the Hsp90 protein isautologous to (derived from) the subject to whom they are administered.The Hsp90 protein and/or antigenic molecules can be purified fromnatural sources, chemically synthesized, or recombinantly produced.Preferably, the complex and/or antigenic molecule is purified inaccordance with a purification method of the present invention disclosedherein above. The invention provides methods for determining doses forhuman cancer immunotherapy by evaluating the optimal dose of an Hsp90protein non-covalently bound to peptide complexes in experimental tumormodels and extrapolating the data. Specifically, a scaling factor notexceeding a fifty-fold increase over the effective dose estimated inanimals, is used as the optimal prescription method for cancerimmunotherapy or vaccination in human subjects. Preferably, the Hsp90protein is GRP94.

[0202] The invention provides combinations of compositions which enhancethe immunocompetence of the host individual and elicit specific immunityagainst infectious agents or specific immunity against preneoplastic andneoplastic cells. The therapeutic regimens and pharmaceuticalcompositions of the invention are described below. These compositionshave the capacity to prevent the onset and progression of infectiousdiseases and prevent the development of tumor cells and to inhibit thegrowth and progression of tumor cells, indicating that such compositionscan induce specific immunity in infectious diseases and cancerimmunotherapy. For example, Hsp90-antigenic molecule complexes can beadministered in combination with other complexes, such as calreticulin,and antigenic molecules in accordance with the methods of the presentinvention.

[0203] Accordingly, the invention provides methods of preventing andtreating cancer in a subject. A representative method comprisesadministering a therapeutically effective amount of an Hsp90 modulator(preferably a GRP94 modulator) to a subject in need thereof. Such asubject can include but is not limited to a subject suffering fromcancer or at risk to develop cancer. Representative modulators that canbe employed in the method comprise ligands that inhibit GRP94 (Hsp90)function. Such ligands are designed and identifed using the screeningmethods disclosed herein and are thus employed as anti-tumor drugs,and/or anti-neoplastic agents. Characterization of these activities canbe accomplished via techniques disclosed herein and known in the art.

[0204] In another embodiment, the method comprises administering acomplex comprising an Hsp90 protein and pertinent antigenic molecule inconjunction with a modulator which stimulates the immunocompetence ofthe host individual and elicits specific immunity against thepreneoplastic and/or neoplastic cells. Preferably, the Hsp90 protein isGRP94.

[0205] As used herein, “preneoplastic” cell refers to a cell which is intransition from a normal to a neoplastic form; and morphologicalevidence, increasingly supported by molecular biologic studies,indicates that preneoplasia progresses through multiple steps.Non-neoplastic cell growth commonly consists of hyperplasia, metaplasia,or most particularly, dysplasia (for review of such abnormal growthconditions. See Robbins & Angell (1976) Basic Pathology, 2d Ed., pp.68-79, W. B. Saunders Co., Philadelphia, Pa.).

[0206] Hyperplasia is a form of controlled cell proliferation involvingan increase in cell number in a tissue or organ, without significantalteration in structure or function. As but one example, endometrialhyperplasia often precedes endometrial cancer. Metaplasia is a form ofcontrolled cell growth in which one type of adult or fullydifferentiated cell substitutes for another type of adult cell.Metaplasia can occur in epithelial or connective tissue cells. Atypicalmetaplasia involves a somewhat disorderly metaplastic epithelium.Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia; it is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplastic cells often haveabnormally large, deeply stained nuclei, and exhibit pleomorphism.Dysplasia characteristically occurs where there exists chronicirritation or inflammation, and is often found in the cervix,respiratory passages, oral cavity, and gall bladder. Althoughpreneoplastic lesions can progress to neoplasia, they can also remainstable for long periods and can even regress, particularly if theinciting agent is removed or if the lesion succumbs to an immunologicalattack by its host.

[0207] The therapeutic regimens and pharmaceutical compositions of theinvention can be used with additional adjuvants or biological responsemodifiers including, but not limited to, the cytokines IFN-α, IFN-γ,IL-2, IL-4, IL-6, TNF, or other cytokine affecting immune cells. Inaccordance with this aspect of the invention, a complex of an Hsp90protein and an antigenic molecule along with a modulator areadministered in combination therapy with one or more of these cytokines.Preferably, the Hsp90 protein is GRP94.

[0208] The invention also pertains to administration of a complex of anHsp90 protein and an antigenic molecule and a modulator to individualsat enhanced risk of cancer due to familial history or environmental riskfactors. Preferably, the Hsp90 protein is GRP94.

[0209] Enzymes in the cells of higher eukaryotes that mediate the steadystate and stress-elicited production of a native GRP94 ligand can alsobe modulated in accordance with the present invention. Particularly,such catabolic enzymes represent appropriate and rational targets formodulation to elicit an increase in the steady state levels of a nativeGRP94 ligand and thereby lead to the elicitation of the structural andfunctional activation of chaperone and peptide binding activity of GRP94disclosed herein.

[0210] Protein misfolding disorders are a common component of numerousgenetic disease states including, but not limited to, cystic fibrosis,familial hypercholesterolemia, retinitis pigmentosa and α1-antitrypsinmisfolding. Compounds that modulate the activity of the Hsp90 family ofmolecular chaperones can thus be used in accordance with a therapeuticmethod of the present invention for reversing the protein foldingdefects that identify the disease state or for enhancing proteintransport from the endoplasmic reticulum of a cell. Thus, a compoundthat modulates the conformation of GRP94 can be used to treat a diseasestate resulting from defects in protein transport into or from theendoplasmic reticulum. Compounds that abrogate GRP94 activity can beused for the treatment of a disease state, such as cancer, wherein atherapeutic benefit can be provided by blocking the egress of proteins(e.g., growth factors) from the endoplasmic reticulum. conversely,compounds that promote GRP94 activity can be used to treat a diseasewherein a therapeutic benefit can be provided by enhancing proteinexport from the endoplasmic reticulum.

[0211] The present invention also pertains to administration ofcompounds for the prevention or amelioration of cellular damage arisingfrom conditions of ischemia/reperfusion including but not limited tocardiac arrest, asystole and sustained ventricular arrythmias, cardiacsurgery, cardiopulmonary bypass surgery, organ transplantation, spinalcord injury, head trauma, stroke, thromboembolic stroke, hemorrhagicstroke, cerebral vasospasm, hypotension, hypoglycemia, statusepilepticus, an epileptic seizure, anxiety, schizophrenia, aneurodegenerative disorder, Alzheimer's disease, Huntington's disease,amyotrophic lateral sclerosis (ALS), or neonatal stress. In oneembodiment, a composition comprising a Hsp90 ligand is administered topromote conformational activation of a Hsp90 protein, thereby promotingits cellular protective function relevant to recovery following a injuryor onset of a disease state associated with ischemia. In anotherembodiment, administration of a composition comprising a Hsp90 ligandcan alter a subsequent cellular response to an ischemic condition at atissue location in a subject. Cells at the tissue location are contactedwith a Hsp90 protein ligand, whereby Hsp90 activity in the cells isenhanced to a degree effective to alter a subsequent cellular responseto an ischemic condition. Preferably, the therapeutic compositioncomprises a ligand identified according to a screening or rational drugdesign method disclosed herein. Also preferably, the therapeuticcomposition modulates the activity of GRP94 or HSP90.

[0212] F.3. Dosage Regimens

[0213] Actual dosage levels of active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to administer anamount of the active compound(s) that is effective to achieve thedesired therapeutic response for a particular subject. The selecteddosage level will depend upon the activity of the particular compound,the route of administration, the severity of the condition beingtreated, and the condition and prior medical history of the subjectbeing treated. However, it is within the skill of the art to start dosesof the compound at levels lower than required to achieve the desiredtherapeutic effect and to gradually increase the dosage until thedesired effect is achieved. If desired, the effective daily dose may bedivided into multiple doses for purposes of administration, e.g., two tofour separate doses per day. It will be understood, however, that thespecific dose level for any particular subject will depend upon avariety of factors including the body weight, general health, diet, timeand route of administration, combination with other drugs and theseverity of the particular disease being treated.

[0214] The dosage ranges for the administration of a modulator dependupon the form of the modulator, and its potency, as described furtherherein, and are amounts large enough to produce the desired effect. Thedosage should not be so large as to cause adverse side effects, such ashyperviscosity syndromes, pulmonary edema, congestive heart failure, andthe like. Generally, the dosage will vary with the age, condition, sexand extent of the disease in the patient and can be determined by one ofskill in the art. The dosage can also be adjusted by the individualphysician in the event of any complication.

[0215] The therapeutic compositions can be administered as a unit dose.The term “unit dose” when used in reference to a therapeutic compositionemployed in the method of the present invention refers to physicallydiscrete units suitable as unitary dosage for the subject, each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect in association with the requireddiluent; i.e., carrier or vehicle.

[0216] The compositions are administered in a manner compatible with thedosage formulation, and in a therapeutically effective amount. Thequantity to be administered depends on the subject to be treated,capacity of the subject's system to utilize the active ingredient, anddegree of therapeutic effect desired. Precise amounts of activeingredient required to be administered depend on the judgment of thepractitioner and are peculiar to each individual. However, suitabledosage ranges for systemic application are disclosed herein and dependon the route of administration. Suitable regimes for administration arealso variable, but are typified by an initial administration followed byrepeated doses at one or more hour intervals by a subsequent injectionor other administration. Alternatively, continuous intravenous infusionsufficient to maintain concentrations in the blood in the rangesspecified for in vivo therapies can also be administered.

[0217] A therapeutically effective amount is an amount of a modulatorsufficient to produce a measurable modulation of Hsp9o protein(preferably GRP94) biological activity in a subject being treated, i.e.,an Hsp90 protein biological activity-modulating amount. Modulation ofHsp90 protein biological activity can be measured using the screeningmethods disclosed herein, via the method disclosed in the Examples, orby other methods known to one skilled in the art.

[0218] The potency of a modulator can vary, and therefore a“therapeutically effective” amount can vary. However, as shown by thepresent assay methods, one skilled in the art can readily assess thepotency and efficacy of a candidate modulator of this invention andadjust the therapeutic regimen accordingly. A modulator of Hsp90 protein(preferably GRP94) biological activity can be evaluated by a variety ofmethods and techniques including the screening assays disclosed herein.

[0219] A preferred modulator has the ability to substantially bind anHsp90 protein in solution at modulator concentrations of less than one(1) micromolar (μM), preferably less than 0.1 μM, and more preferablyless than 0.01 μM. By “substantially” is meant that at least a 50percent reduction in biological activity is observed by modulation inthe presence of the modulator, and at 50% reduction is referred toherein as an “IC50 value”.

[0220] In one embodiment, the therapeutically effective amount of amodulator can respectively range from about 0.01 mg to about 10,000 mgper day. Alternatively, the therapeutically effective amount of amodulator can respectively range from about 0.1 mg to about 1,000 mg perday. Alternatively, the therapeutically effective amount of a modulatorcan respectively range from about 1 mg to about 300 mg per day. In apreferred embodiment, the therapeutically effective amount of amodulator can respectively range from about 15 mg per kg body weight perday to about 35 mg per kg body weight per day.

[0221] It was established in experimental tumor models (Blachere et al.,1993) that the lowest dose of heat shock proteins noncovalently bound topeptide complexes which produced tumor regression in mice was between 10and 25 microgram/mouse weighing 20-25 g which is equal to 25 mg/25 g=1mg/kg. Conventional methods extrapolate to human dosages based on bodyweight and surface area. For example, conventional methods ofextrapolating human dosage based on body weight can be carried out asfollows: since the conversion factor for converting the mouse dosage tohuman dosage is Dose Human per kg=Dose Mouse per kg×12 (Freireich et al.(1966) Cancer Chemotherap Rep 50:219-244), the effective dose ofHsp90-peptide complexes in humans weighing 70 kg should be 1mg/kg÷12×70, i.e., about 6 mg (5.8 mg).

[0222] Drug doses are also given in milligrams per square meter of bodysurface area because this method rather than body weight achieves a goodcorrelation to certain metabolic and excretionary functions (Shirkey(1965) JAMA 193:443). Moreover, body surface area can be used as acommon denominator for drug dosage in adults and children as well as indifferent animal species as described by Freireich et al. (1966) CancerChemotherap Rep 50:219-244. Briefly, to express a mg/kg dose in anygiven species as the equivalent mg/sq m dose, multiply the dose by theappropriate km factor. In adult human, 100 mg/kg is equivalent to 100mg/kg×37 kg/sq m=3700 mg/sq m.

[0223] International Publication Nos. WO 95/24923, WO 97/10000, WO97/10002, and WO 98/34641, as well as U.S. Pat. Nos. 5,750,119,5,830,464, and 5,837,251, each provide dosages of the purified complexesof heat shock proteins and antigenic molecules, and the entire contentsof each of these documents are herein incorporated by reference.Briefly, and as applied to the present invention, an amount of Hsp90protein (preferably GRP94)-antigenic molecule complexes is administeredthat is in the range of about 10 microgram to about 600 micrograms for ahuman subject, the preferred human dosage being the same as used in a 25g mouse, i.e., in the range of 10-100 micrograms. The dosage for Hsp90protein (preferably GRP94)-peptide complexes in a human subject providedby the present invention is in the range of about 50 to 5,000micrograms, the preferred dosage being 100 micrograms.

[0224] In a series of preferred and more preferred embodiments, theHsp90-peptide complex is administered in an amount of less than about 50micrograms. In this case, the Hsp90 protein (preferably GRP94)-peptidecomplex is preferably administered in an amount of ranging from about 5to about 49 micrograms. In a preferred embodiment, a GRP94-peptidecomplex is administered in an amount of less than about 10 micrograms.In this case, the GRP94-peptide complex is preferably administered in anamount ranging from about 0.1 to about 9.0 micrograms. More preferably,the GRP94-peptide complexes is administered in an amount ranging fromabout 0.5 to about 2.0 micrograms. In accordance with one aspect of thepresent invention, administration of a lower dosage of complex isfacilitated and preferred when a modulator is also administered.

[0225] The doses recited above are preferably given once weekly for aperiod of about 4-6 weeks, and the mode or site of administration ispreferably varied with each administration. In a preferred example,subcutaneous administrations are given, with each site of administrationvaried sequentially. For example, half the dose can be given in one siteand the other half on an other site on the same day.

[0226] Alternatively, the mode of administration is sequentially varied.For example, weekly injections are given in sequence subcutaneously,intramuscularly, intravenously or intraperitoneally. After 4-6 weeks,further injections are preferably given at two-week intervals over aperiod of time of one month. Later injections can be given monthly. Thepace of later injections can be modified, depending upon the subject'sclinical progress and responsiveness to the immunotherapy.

[0227] F.4. Therapeutic Compositions for Immune Responses to Cancer

[0228] Compositions comprising an Hsp90 protein bound (e.g.,GRP94-preferably non-covalently bound) to antigenic molecules areadministered to elicit an effective specific immune response to thecomplexed antigenic molecules (and preferably not to the HSP90 protein).In a preferred embodiment, non-covalent complexes of the Hsp90 proteinwith peptides are prepared and purified postoperatively from tumor cellsobtained from the cancer patient that have also been treated with anHsp90 protein biological activity modulator in accordance with thepresent invention. A preferred Hsp90 protein is GRP94. In a morepreferred embodiment, the complexes are purified using an affinitypurification method of the present invention, as disclosed herein above.

[0229] In accordance with the methods described herein, immunogenic orantigenic peptides that are endogenously complexed to Hsp90 (e.g. GRP94)or MHC antigens can be used as antigenic molecules. For example, suchpeptides can be prepared that stimulate cytotoxic T cell responsesagainst different tumor antigens (e.g., tyrosinase, gp100, melan-A,gp75, mucins, etc.) and viral proteins including, but not limited to,proteins of immunodeficiency virus type I (HIV-I), humanimmunodeficiency virus type II (HIV-II), hepatitis type A, hepatitistype B, hepatitis type C, influenza, varicella, adenovirus, herpessimplex type I (HSV-I), herpes simplex type II (HSV-II), rinderpest,rhinovirus, echovirus, rotavirus, respiratory syncytial virus (RSV),papilloma virus, papova virus, cytomegalovirus, echinovirus, arbovirus,huntavirus, coxsackie virus, mumps virus, measles virus, rubella virusand polio virus. In the embodiment wherein the antigenic molecules arepeptides noncovalently complexed to GRP94 in vivo, the complexes can beisolated from cells, or alternatively, produced in vitro from purifiedpreparations each of GRP94 and antigenic molecules. The complexes can befurther purified using an affinity purification method of the presentinvention, as disclosed herein above.

[0230] In another specific embodiment, antigens of cancers (e.g.,tumors) or infectious agents (e.g., viral antigen, bacterial antigens,etc.) can be obtained by purification from natural sources, by chemicalsynthesis, or recombinantly, and, through in vitro procedures such asthose described herein, complexed to GRP94. The complexes can also befurther purified using an affinity purification method of the presentinvention, as disclosed herein above.

[0231] F.5. Formulation

[0232] In accordance with the present invention, modulators as well asantigenic molecule complexes can be formulated into pharmaceuticalpreparations for administration to a subject for treatment or preventionof cancer or infectious diseases. Compositions comprising a complexprepared in accordance with the present invention are formulated in acompatible pharmaceutical carrier can be prepared, packaged, and labeledfor treatment of the indicated disorder (e.g. cancer or infectiousdisease).

[0233] If the modulator or complex is water-soluble, then it can beformulated in an appropriate buffer, for example, phosphate bufferedsaline or other physiologically compatible solutions. Alternatively, ifa modulator or a resulting complex has poor solubility in aqueoussolvents, then it can be formulated with a non-ionic surfactant, such asTWEEN™, or polyethylene glycol. Thus, the compounds and theirphysiologically acceptable solvates can be formulated for administrationby inhalation or insufflation (either through the mouth or the nose) ororal, buccal, parenteral, rectal administration or, in the case oftumors, directly injected into a solid tumor.

[0234] For oral administration, the pharmaceutical preparation can be inliquid form, for example, solutions, syrups or suspensions, or can bepresented as a drug product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations can be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, orfractionated vegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The pharmaceuticalcompositions can take the form of, for example, tablets or capsulesprepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (e.g., pregelatinized maize starch,polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,lactose, microcrystalline cellulose or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc or silica); disintegrants(e.g., potato starch or sodium starch glycolate); or wetting agents(e.g., sodium lauryl sulphate). The tablets can be coated by methodswell-known in the art. Preparations for oral administration can besuitably formulated to give controlled release of the active compound.

[0235] For buccal administration, the compositions can take the form oftablets or lozenges formulated in conventional manner. Foradministration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitcan be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, for example, gelatin for use in an inhaleror insufflator can be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

[0236] The compositions can be formulated for parenteral administrationby injection, e.g., by bolus injection or continuous infusion.Formulations for injection can be presented in unit dosage form, forexample, in ampules or in multi-dose containers, with an addedpreservative. The compositions can take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient can be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

[0237] The compounds can also be formulated in rectal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

[0238] In addition to the formulations described previously, thecompounds can also be formulated as a depot preparation. Such longacting formulations can be administered by implantation (for example,subcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the compounds can be formulated with suitable polymeric orhydrophobic materials (for example, as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt. Liposomes and emulsions are wellknown examples of delivery vehicles or carriers for hydrophilic drugs.

[0239] The compositions can, if desired, be presented in a pack ordispenser device which can contain one or more unit dosage formscontaining the active ingredient. The pack can for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice can be accompanied by instructions for administration.

[0240] The invention also provides kits for carrying out the therapeuticregimens of the invention. Such kits comprise in one or more containerstherapeutically or prophylactically effective amounts of a modulatorand/or a antigenic molecule complex in pharmaceutically acceptable form.The modulator and the antigenic molecule complex in a vial of a kit ofthe invention can be in the form of a pharmaceutically acceptablesolution, e.g., in combination with sterile saline, dextrose solution,or buffered solution, or other pharmaceutically acceptable sterilefluid. Alternatively, the modulator or complex can be lyophilized ordesiccated; in this instance, the kit optionally further comprises in acontainer a pharmaceutically acceptable solution (e.g., saline, dextrosesolution, etc.), preferably sterile, to reconstitute the modulatorcomplex to form a solution for injection purposes.

[0241] In another embodiment, a kit of the invention further comprisesneedles or syringes, preferably packaged in sterile form, for injectingthe modulator and complex, and/or a packaged alcohol pad. Instructionsare optionally included for administration of antigenic moleculecomplexes by a clinician or by the subject.

[0242] G. Target Infectious Diseases

[0243] Infectious diseases that can be treated or prevented by themethods of the present invention are caused by infectious agentsincluding, but not limited to, viruses, bacteria, fungi, protozoa andparasites. In one embodiment of the present invention wherein it isdesired to treat a subject having an infectious disease, theabove-described affinity purification methods are used to isolateGRP94-peptide complexes from cells infected with an infectious organism,e.g., of a cell line or from a subject.

[0244] Viral diseases that can be treated or prevented by the methods ofthe present invention include, but are not limited to, those caused byhepatitis type A, hepatitis type B, hepatitis type C, influenza,varicella, adenovirus, herpes simplex type I (HSV-I), herpes simplextype II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus,respiratory syncytial virus (RSV), papilloma virus, papova virus,cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus,mumps virus, measles virus, rubella virus, polio virus, humanimmunodeficiency virus type I (HIV-I), and human immunodeficiency virustype II (HIV-II).

[0245] Bacterial diseases that can be treated or prevented by themethods of the present invention are caused by bacteria including, butnot limited to, Mycobacteria, Mycoplasma, Neisseria, and Legionella.

[0246] Protozoal diseases that can be treated or prevented by themethods of the present invention are caused by protozoa including, butnot limited to, Leishmania, Kokzidioa, and Trypanosoma. Parasiticdiseases that can be treated or prevented by the methods of the presentinvention are caused by parasites including, but not limited to,Chlamydia and Rickettsia.

[0247] H. Target Cancers

[0248] Cancers that can be treated or prevented by the methods of thepresent invention include, but not limited to human sarcomas andcarcinomas, including but not limited to fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acutemyelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic,monocytic and erythroleukemia); chronic leukemia (chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia); andpolycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin'sdisease), multiple myeloma, Waldenströom's macroglobulinemia, and heavychain disease.

[0249] In a specific embodiment the cancer is metastatic. In anotherspecific embodiment, the subject having a cancer is immunosuppressed byreason of having undergone anti-cancer therapy (e.g., chemotherapyradiation) prior to administration of the GRP94-antigenic moleculecomplexes and a GRP94 modulator in accordance with the presentinvention.

[0250] I. Combination With Adoptive Immunotherapy

[0251] Adoptive immunotherapy refers to a therapeutic approach fortreating cancer or infectious diseases in which immune cells areadministered to a host with the aim that the cells mediate eitherdirectly or indirectly specific immunity to tumor cells and/or antigeniccomponents or regression of the tumor or treatment of infectiousdiseases, as the case can be. In accordance with the methods describedherein, APC are sensitized with GRP94 preferably noncovalently complexedwith antigenic (or immunogenic) molecules in conjunction with a GRP94biological activity modulator and used in adoptive immunotherapy.

[0252] According to one embodiment of the present invention, therapy byadministration of GRP94-peptide complexes and a GRP94 biologicalactivity modulator, using any desired route of administration, iscombined with adoptive immunotherapy using APC sensitized withGRP94-antigenic molecule complexes and a modulator. The sensitized APCcan be administered concurrently with GRP94-peptide complexes and themodulator, or before or after administration of GRP94-peptide complexesand the modulator. Furthermore, the mode of administration can bevaried, including but not limited to, e.g., subcutaneously,intravenously, intraperitoneally, intramuscularly, intradermally ormucosally.

[0253] I.1. Obtaining Macrophages and Antigen-Presenting Cells

[0254] The antigen-presenting cells, including but not limited tomacrophages, dendritic cells and B-cells, are preferably obtained byproduction in vitro from stem and progenitor cells from human peripheralblood or bone marrow as described by Inaba (1992) J Exp Med176:1693-1702.

[0255] APC can be obtained by any of various methods known in the art.In a preferred aspect human macrophages are used, obtained from humanblood cells. By way of example but not limitation, macrophages can beobtained as follows: mononuclear cells are isolated from peripheralblood of a subject (preferably the subject to be treated), byFicoll-Hypaque gradient centrifugation and are seeded on tissue culturedishes which are pre-coated with the subject's own serum or with otherAB+ human serum. The cells are incubated at 37° C. for 1 hr, thennon-adherent cells are removed by pipetting. To the adherent cells leftin the dish, is added cold (4° C.) 1 mM EDTA in phosphate-bufferedsaline and the dishes are left at room temperature for 15 minutes. Thecells are harvested, washed with RPMI buffer and suspended in RPMIbuffer. Increased numbers of macrophages can be obtained by incubatingat 37° C. with macrophage-colony stimulating factor (M-CSF); increasednumbers of dendritic cells can be obtained by incubating withgranulocyte-macrophage-colony stimulating factor (GM-CSF) as describedin detail by Inaba, et al. (1992).

[0256] I.2. Sensitization of Macrophages and Antigen Presenting CellsWith GRP94-Peptide Complexes

[0257] APC are sensitized with GRP94 (preferably noncovalently) bound toantigenic molecules by incubating the cells in vitro with the complexesand a modulator. The APC are sensitized with complexes of GRP94 andantigenic molecules preferably by incubating in vitro with theGRP94-complex and a modulator at 37° C. for 15 minutes to 24 hours. Byway of example but not limitation, 4×10⁷ macrophages can be incubatedwith 10 microgram GRP94-peptide complexes per ml or 100 microgramGRP94-peptide complexes per mL and a modulator in an equimolar amountwith respect to the GRP94-peptide complex at 37° C. for 15 minutes-24hours in 1 mL plain RPMI medium. The cells are washed three times andresuspended in a physiological medium preferably sterile, at aconvenient concentration (e.g., 1×10⁷ /ml) for injection in a subject.Preferably, the subject into which the sensitized APCs are injected isthe subject from which the APC were originally isolated (autologousembodiment).

[0258] Optionally, the ability of sensitized APC to stimulate, forexample, the antigen-specific, class I-restricted cytotoxicT-lymphocytes (CTL) can be monitored by their ability to stimulate CTLsto release tumor necrosis factor, and by their ability to act as targetsof such CTLs.

[0259] I.3. Reinfusion of Sensitized APC

[0260] The sensitized APC are reinfused into the subject systemically,preferably intravenously, by conventional clinical procedures. Theseactivated cells are reinfused, preferentially by systemic administrationinto the autologous subject. Subjects generally receive from about 10⁶to about 10¹² sensitized macrophages, depending on the condition of thesubject. In some regimens, subjects can optionally receive in addition asuitable dosage of a biological response modifier including but notlimited to the cytokines IFN-α, IFN-γ, IL-2, IL-4, IL-6, TNF or othercytokine growth factor.

[0261] J. Autologous Embodiment

[0262] The specific immunogenicity of an Hsp90 protein derives not fromHsp90 protein per se, but from the peptides bound to them. In apreferred embodiment of the invention directed to the use of autologouscomplexes of GRP94-peptides as cancer vaccines wherein theimmunogenicity has been enhanced with a modulator in accordance with thepresent invention, two of the most intractable hurdles to cancerimmunotherapy are circumvented. First is the possibility that humancancers, like cancers of experimental animals, are antigenicallydistinct. Thus, in an embodiment of the present invention, GRP94chaperones antigenic peptides of the cancer cells from which they arederived and circumvent this hurdle.

[0263] Second, most current approaches to cancer immunotherapy focus ondetermining the CTL-recognized epitopes of cancer cell lines. Thisapproach requires the availability of cell lines and CTLs againstcancers. These reagents are unavailable for an overwhelming proportionof human cancers. Thus, in an embodiment of the present inventiondirected to autologous complexes of GRP94 and peptides, preferablywherein the immunogenicity has been enhanced with a modulator of thepresent invention, cancer immunotherapy does not depend on theavailability of cell lines or CTLs nor does it require definition of theantigenic epitopes of cancer cells. These advantages make autologousHsp90 proteins (e.g., GRP94) noncovalently bound to peptide complexesattractive and novel immunogens against cancer.

[0264] K. Prevention and Treatment of Primary and Metastatic NeoplasticDiseases

[0265] There are many reasons why immunotherapy as provided by thepresent invention is desired for use in cancer patients. First, ifcancer patients are immunosuppressed and surgery, with anesthesia, andsubsequent chemotherapy, can worsen the immunosuppression, then withappropriate immunotherapy in the preoperative period, thisimmunosuppression can be prevented or reversed. This could lead to fewerinfectious complications and to accelerated wound healing. Second, tumorbulk is minimal following surgery and immunotherapy is most likely to beeffective in this situation. A third reason is the possibility thattumor cells are shed into the circulation at surgery and effectiveimmunotherapy applied at this time can eliminate these cells.

[0266] The preventive and therapeutic methods of the invention aredirected at enhancing the immunocompetence of the cancer patient eitherbefore surgery, at or after surgery, and to induce tumor-specificimmunity to cancer cells, with the objective being inhibition of cancer,and with the ultimate clinical objective being total cancer regressionand eradication.

[0267] L. Monitoring of Effects During Cancer Prevention andImmunotherapy with Hsp90 Protein-Antigenic Molecule Complexes

[0268] The effect of immunotherapy with GRP94-antigenic moleculecomplexes on development and progression of neoplastic diseases can bemonitored by any methods known to one skilled in the art, including butnot limited to measuring: 1) delayed hypersensitivity as an assessmentof cellular immunity; 2) activity of cytolytic T-lymphocytes in vitro;3) levels of tumor specific antigens, e.g., carcinoembryonic (CEA)antigens; 4) changes in the morphology of tumors using techniques suchas a computed tomographic (CT) scan; 5) changes in levels of putativebiomarkers of risk for a particular cancer in individuals at high risk,and 6) changes in the morphology of tumors using a sonogram.

[0269] Delayed Hypersensitivity Skin Test. Delayed hypersensitivity skintests are of great value in the overall immunocompetence and cellularimmunity to an antigen. Inability to react to a battery of common skinantigens is termed anergy (Sato et al. (1995) Clin Immunol Pathol74:35-43). Proper technique of skin testing requires that the antigensbe stored sterile at 4° C., protected from light and reconstitutedshortly before use. A 25- or 27-gauge needle ensures intradermal, ratherthan subcutaneous, administration of antigen. Twenty-four andforty-eight hours after intradermal administration of the antigen, thelargest dimensions of both erythema and induration are measured with aruler. Hypoactivity to any given antigen or group of antigens isconfirmed by testing with higher concentrations of antigen or, inambiguous circumstances, by a repeat test with an intermediateconcentration.

[0270] Activity of Cytolytic T-lymphocytes In vitro. 8×10⁶ peripheralblood derived T lymphocytes isolated by the Ficoll-Hypaquecentrifugation gradient technique, are restimulated with 4×10⁴ mitomycinC treated tumor cells in 3 ml RPMI medium containing 10% fetal calfserum. In some experiments, 33% secondary mixed lymphocyte culturesupernatant or IL-2, is included in the culture medium as a source of Tcell growth factors.

[0271] In order to measure the primary response of cytolyticT-lymphocytes after immunization, T cells are cultured without thestimulator tumor cells. In other experiments, T cells are restimulatedwith antigenically distinct cells. After six days, the cultures aretested for cytotoxicity in a 4 hour ⁵¹Cr-release assay. The spontaneous⁵¹Cr-release of the targets should reach a level less than 20%. For theanti-MHC class I blocking activity, a tenfold concentrated supernatantof W6/32 hybridoma is added to the test at a final concentration ofabout 12.5% (Heike et al. (1994) J Immunotherapy 15:165-174).

[0272] Levels of Tumor Specific Antigens. Although it can not bepossible to detect unique tumor antigens on all tumors, many tumorsdisplay antigens that distinguish them from normal cells. Monoclonalantibody reagents have permitted the isolation and biochemicalcharacterization of the antigens and have been invaluable diagnosticallyfor distinction of transformed from nontransformed cells and fordefinition of the cell lineage of transformed cells. Thebest-characterized human tumor-associated antigens are the oncofetalantigens. These antigens are expressed during embryogenesis, but areabsent or very difficult to detect in normal adult tissue. The prototypeantigen is carcinoembryonic antigen (CEA), a glycoprotein found on fetalgut an human colon cancer cells, but not on normal adult colon cells.Since CEA is shed from colon carcinoma cells and found in the serum, itwas originally thought that the presence of this antigen in the serumcould be used to screen subjects for colon cancer. However, subjectswith other tumors, such as pancreatic and breast cancer, also haveelevated serum levels of CEA. Therefore, monitoring the fall and rise ofCEA levels in cancer patients undergoing therapy has proven useful forpredicting tumor progression and responses to treatment.

[0273] Several other oncofetal antigens have been useful for diagnosingand monitoring human tumors, e.g., alpha-fetoprotein, an alpha-globulinnormally secreted by fetal liver and yolk sac cells, is found in theserum of subjects with liver and germinal cell tumors and can be used asa matter of disease status.

[0274] Computed Tomographic (CT) Scan. CT remains the choice oftechniques for the accurate staging of cancers. CT has proved moresensitive and specific than any other imaging techniques for thedetection of metastases.

[0275] Measurement of Putative Biomarkers. The levels of a putativebiomarker for risk of a specific cancer are measured to monitor theeffect of GRP94 noncovalently bound to peptide complexes. For example,in individuals at enhanced risk for prostate cancer, serumprostate-specific antigen (PSA) is measured by the procedure describedby Brawer et al. (1992) J Urol 147:841-845 and Catalona et al. (1993)JAMA 270:948-958; or in individuals at risk for colorectal cancer CEA ismeasured as described above; and in individuals at enhanced risk forbreast cancer, 16-α-hydroxylation of estradiol is measured by theprocedure described by Schneider et al. (1982) Proc Natl Acad Sci USA79:3047-3051. The references cited above are incorporated by referenceherein in their entirety.

[0276] Sonogram. A Sonogram remains an alternative choice of techniquefor the accurate staging of cancers.

[0277] M. Target Disorders/Traumas Associated with Ischemia

[0278] The present invention provides methods for treating andpreventing ischemia-induced damage comprising administering a Hsp90protein modulator to a subject wherein Hsp90 activity modulation isdesired. The term “ischemia”, as used herein, is a loss of blood flow toa tissue. Blood loss is characterized by deprivation of both oxygen andglucose, and leads to ischemic necrosis or infarction. Thus, the term“ischemia” refers to both conditions of oxygen deprivation and ofnutrient deprivation. Loss of blood flow to a particular vascular regionis described as “focal ischemia”. Loss of blood flow to an entire tissueor body is referred to as “global ischemia”.

[0279] The present invention provides therapeutic compositions andmethods to ameliorate cellular damage arising from conditions ofischemia/reperfusion including but not limited to cardiac arrest,asystole and sustained ventricular arrythmias, cardiac surgery,cardiopulmonary bypass surgery, organ transplantation, spinal cordinjury, head trauma, stroke, thromboembolic stroke, hemorrhagic stroke,cerebral vasospasm, hypotension, hypoglycemia, status epilepticus, anepileptic seizure, anxiety, schizophrenia, a neurodegenerative disorder,Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis(ALS), neonatal stress, and any condition in which a neuroprotectantcomposition that prevents or ameliorates ischemic cerebral damage isindicated, useful, recommended, or prescribed.

[0280] The destructive effects of ischemia/reperfusion are manifest as acascade of deleterious events that lead to cell death and ultimatelyorgan failure. The metabolic events underlying ischemia-induced celldeath include energy failure through ATP depletion, cellular acidosis,glutamate release, calcium ion influx, stimulation of membranephospholipid degradation and subsequent free-fatty-acid accumulation,and free radical degeneration. Further, in contrast to apoptotic celldeath, ischemia-induced cell death is characterized by degeneration ofthe most distal cell regions, and subsequent progressive degeneration ofthe cell soma and nucleus (Yamamoto et al. (1986) Brain Res 384:1-10;Yamamoto et al. (1990) Acta Neuropathol 80:487-492). Consistent withthis degeneration profile, cells that bear extended processes, such asneuronal cells, are particularly sensitive to ischemic damage. Althoughnot intended to be limited according to any particular theory, theseobservations suggest that intracellular transport and proteinavailability are essential components of cellular response to stress,and further implicate molecular components of such function, includingHsp90 proteins, as targets for ischemic response.

[0281] Thus, in one embodiment, the present invention pertains to thetreatment of central nervous system ischemia. Examples of centralnervous system ischemia include cerebral ischemic and spinal columnischemia. “Cerebral ischemia” is the interruption or reduction of bloodflow in the arteries in or leading to the brain, usually as a result ofa blood clot (thrombus) or other matter (embolus) occluding the artery.

[0282] A therapeutic composition of the present invention for theprevention or amelioration of ischemia-induced damage comprises a Hsp90protein ligand. Preferably, such modulators promote or stabilize anactive structural conformation of an endogenous Hsp90 protein. Alsopreferably, the Hsp90 ligand modulates the activity of GRP94 or HSP90.Desired properties of a composition having a cellular protectant effectinclude the following: (1) easy administration by oral or injectableroutes (e.g., not significantly degraded in the stomach, intestine, orvascular system such that it reaches the tissues to be treated in atherapeutically effective amount), (2) therapeutic activity (e.g.,efficacy) when administered following an ischemic insult, and (3)minimal or no side effects including impairment of cognition, disruptionof motor performance, sedation, hyperexcitability, neuronalvacuolization, and impaired cardiovascular activity.

[0283] Compositions comprising Hsp90 protein ligands can be administeredimmediately following a trauma or other event that induces an ischemiccondition. Alternatively, such a composition may be administeredcontinuously or intermittently following detection of a progressivedisorder, including but not limited to neurodegenerative diseases. Instill another embodiment, such a composition may be administered toprevent or improve recovery from a subsequent ischemic condition. Ineach case, effective dose and administration profiles can be determinedusing standard experiments directed at such determination in animalmodels of ischemic conditions as disclosed in, for example, Tacchini etal. (1997) Hepatology 26(1):186-191 and U.S. Pat. Nos. 4,968,671,5,504,090, and 5,733,916. Exemplary animal models are described hereinbelow.

[0284] In another embodiment, the present invention pertains totreatment of tissue prior to transplantation. Such tissue is entirelydevascularized following removal from the donor body. A therapeuticcomposition comprising a Hsp90 protein ligand can promote recovery andhealth of the transplanted tissue. Several methods for providing such acompound to donor or transplanted tissue are known in the art, includingbut not limited to administering the therapeutic compound that promotesorgan preservation and health to a donor subject prior to procurance ofthe organ, perfusing an isolated organ with the therapeutic composition,and administering the composition to a transplant recipient prior,concurrent, or following tissue transplantation. See Mizoe et al. (1997)J Surg Res 73(2):160-165 and U.S. Pat. Nos. 5,066,578; 5,756,492; and6,080,730.

[0285] In still another embodiment, a composition comprising a Hsp90protein modulator can be repititiously provided to a subject in theabsence of an ischemic condition, whereby the ability of the subject totolerate a subsequent ischemic condition is enhanced. Therapeuticcompositions comprising a Hsp90 ligand of the present invention canprovide such a cellular protectant effect. Preferably, a dose of thetherapeutic composition intended to induce ischemic tolerance wouldeffect a mild ischemic condition as disclosed, for example, in Chen etal. (1996) J Cereb Blood Flow Metab 16:566-577 and U.S. Pat. Nos.5,504,090 and 5,733,916.

[0286] M.1. In vivo Models of Ischemia

[0287] Numerous models of ischemic injury and disease are available forevaluating the therapeutic capacity of compositions comprising Hsp90protein modulators. In addition to animal models described herein below,see also Massa et al. (1996) “The Stress Gene Response in Brain” inCerebrovascular and Brain Metabolism Reviews, pp. 95-158,Lippincott-Raven Publishers, Philadelphia, Pa. and references citedtherein. One skilled in the art will appreciate that alternative modelscan be used as disclosed. To assess therapeutic capacity, candidatecompounds can be administered, for example, as a single dose givenintraperitoneally immediately or 30 minutes after reinstating bloodflow.

[0288] Transient Global Cerebral Ischemia. U.S. Pat. No. 5,571,840discloses a dog model of cardiac arrest. According to this model, adultdogs are anesthetised and mechanically ventilated to maintain surgicalanesthesia and suppression of corneal reflexes. Expired CO₂ tension andesophageal temperature are stably maintained before arrest and for atleast one hour after resuscitation. Two venous catheters are inserted;one passed by way of the left external jugular vein to the right atriumfor administration of resuscitation drugs, and the other into a muscularbranch of the left femoral vein for fluid administration. Arterial bloodpressure is measured through a catheter placed in a muscular branch ofthe left femoral vein for fluid administration. Arterial blood pressureis measured through a catheter placed in a muscular branch of the leftfemoral artery. Subcutaneous disk electrodes are placed to monitor anelectrocardiogram (ECG).

[0289] Each animal is intravenously hydrated before arrest and duringrecovery. All catheters and electrical leads are passed subcutaneouslyto exit the skin in the dorsal midscapular region for later attachmentto a dog jacket and hydraulic/electric swivel. Pulsatile and meanarterial blood pressure (MAP), ECG, and end-expiratory CO₂ can becontinuously recorded on a six-channel oscillograph. At the conclusionof surgical instrumentation, anesthesia is discontinued and ventilationproceeds with room air. When corneal reflexes are apparent, the heart isfibrillated by delivering a 10-15 second, 60 Hz, 2 msec square-wavestimulus to the left ventricular epicardium. Ventilation is discontinuedand circulatory arrest is confirmed by ECG, MAP, and direct observationof the heart. After 9 minutes of normothermic ventricular fibrillation,ventilation is restored and direct cardiac massage is maintained MAPabove 75 mmHg. Mechanical ventilation is continued until spontaneousventilation ensues, but for not longer than 6 hours (typically only 30minutes).

[0290] Conditions of stroke can be approximated by occlusion of theprimary arteries to the brain. In one model, a bilateral common carotidartery occlusion is performed in the gerbil as further disclosed inKarpiak et al. (1989) Ann Rev Pharmacol Toxicol 29:403, Ginsberg & Busto(1989) Stroke 20:1627, and U.S. Pat. No. 6,017,965. Briefly, blood flowto the brain is interrupted for 7 minutes by clamping the carotidarteries. During the course of these experiments, the core bodytemperature of the animals is maintained at 37° C. to prevent ahypothermic reaction.

[0291] Permanent Focal Cerebral Ischemia. In another model of cerebralischemia, the middle cerebral artery is occluded in rat as disclosed inKarpiak et al. (1989) Ann Rev Pharmacol Toxicol 29:403, Ginsberg & Busto(1989) Stroke 20:1627, Chen et al. (1996) Mol Endocrinol 10:682-693, andU.S. Pat. No. 6,017,965. According to this model, the middle cerebralartery is permanently occluded by passing a small piece of suture threadthrough the carotid artery to the region of the middle cerebral artery.Core body temperature is maintained at 37° C. This model is differentfrom the bilateral common carotid artery occlusion in gerbil ineliciting a more restricted brain infarct, and thereby approximating adifferent kind of stroke (focal thrombotic stroke).

[0292] Transient Focal Cerebral Ischemia. In another model of focalcerebral ischemia in the rat, the middle cerebral artery is temporarilyoccluded by passing a small piece of suture thread through the carotidartery to the region of the middle cerebral artery. The suture thread iswithdrawn after an ischemic period of 2 hours. Core body temperature ismaintained at 37° C.

[0293] Additional models of focal ischemia include, but are not limitedto, photochemically induced focal cerebral thrombosis, blood clotembolization, microsphere embolization and related methods. See McAuley(1995) Cerebrovasc Brain Metab Review 7:153-180.

[0294] Renal Ischemia. Adult male rats are anesthetized withphenobarbital (50 mg/kg) and the body temperature of rats is maintainedbetween 36-37° C. Renal ischemia is induced by clamping the left renalartery for 15 minutes (mild ischemia) or 45 minutes (severe ischemia),followed by reperfusion for 5 hours, as disclosed in Kuznetsov (1996)Proc Natl Acad Sci USA 93:8584-8589.

[0295] M.2. In vitro Models of Ischemia

[0296] Cell Culture Model of Epithelial Ischemia. Canine kidney (MDCK)cells are grown in Dulbecco's minimal essential medium supplemented with5% fetal bovine serum. Rat thyroid (PCC13) cells are grown in Coon'smodified Ham's F-12 medium (Sigma of St. Louis, Mo.) supplemented with5% bovine calf serum and a hormone mixture as described in Grollman etal. (1993) J Biol Chem 268:3604-3609. Cultured MDCK or PCC13 cells aresubjected to inhibition of oxidative metabolism by treatment withantimycin A, a specific inhibitor of mitochondrial oxidativephosphorylation as disclosed in Ramachandran & Gottlieb (1961) BiochimBiophys Acta 53:396-402. Alternatively, or in addition, the cells can betreated with 2-deoxyglucose, a nonhydrolyzble analog of glucose, toinhibit glycolytic metabolism. See Bacalloa et al. (1994) J Cell Sci107:3301-3313, Mandel et al. (1994) J Cell Sci 107:3315-224, andKuznetsov (1996) Proc Natl Acad Sci USA 93:8584-8589.

[0297] Cell Culture Model of Oxygen and Glucose Deprivation. Chinesehamster ovary (CHO) cells are grown in Ham's F-10 medium containing 15%newborn calf serum (GibcoBRL of Gaithersburg, Md.). Cells (5 ml) areseeded at a density of 150,000 cells per ml to T25 flasks (Corning ofActon, Mass.) and are used for experiments in a subconfluent stateapproximately 48 hours later. To achieve glucose deprivation, 15% serumis added to F-10 medium prepared without glucose, resulting in apartially glucose deficient broth. During incubation, cells use theremaining glucose after about 20 hours, as can be determined using aSigma glucose colorimetric assay kit. Glucose-deprived cells areharvested after an additional 24 hours of incubation.

[0298] To achieve anoxia, cultures in fell medium (or in full mediumcontaining 50% additional glucose) were placed in a sealed Brewer jar(Baltimore Biological Laboratory, Microbiology Systems of Baltimore,Md.) and anaerobiosis was initiated by using a hydrogen generator in a4-7% carbon dioxide atmosphere as described previously by Anderson &Matovcik (1977) Science 197:1371-1374 and Seip & Evans (1980) J ClinMicrobiol 11:226-233. The oxygen concentration in the jar is decreasedto <0.4% in 100 minutes, and the concentration of oxygen at cell depthin a nonagitated solution is calculated to be within 1% of theenvironmental value within 30 minutes. Such a calculation can be madeaccording to the methods described in Gerweck et al. (1979) Cancer Res39:966-972. The formation of water vapor from hydrogen and oxygen causesa brief (about 15 minute) temperature increase to about 38.6° C. in theculture medium soon after initiation of anaerobiosis. This increase isinsufficient to elicit a heat-shock response.

[0299] Anoxia can be verified using a methylene blue indicator solution.This solution becomes colorless (indicating the absence of oxygen) 5-6hours after the initiation of anaerobiosis. A constant glucoseconcentration (1 g/L) can be maintained by changing the medium at 24hours prior to and immediately prior to the initiation of anaerobiosis.

[0300] Cell Culture Model of Cerebral Ischemia. Isolated neurons can becultured on a monolayer comprising a growth-permissive substrate, suchas an immobilized monolayer of a purified, growth-promoting factor, sucha monolayer comprising collagen, fibronectin, of the L1 glycoprotein. Asan exemplary procedure, neurons (post-natal days 2-7) are dissociated bytrypsinization essentially as described, for example, in U.S. Pat. No.5,932,542. Neurons are added to a well coated with a growth-promotingfactor, followed by addition of either a single concentration orincreasing concentrations of the candidate composition. Neurons arecultured overnight (about 16 hours) at 37° C., and then neuriteoutgrowth is measured. Hypoxia/anoxia can be achieved as describedherein above. Neurite outgrowth of cells subjected to ischemicconditions and to which a candidate therapeutic composition wasadministered can then be compared to neurite outgrowth on control cellsalso subjected to ischemic conditions without administration of atherapeutic composition.

[0301] Cell Culture Model of Glutamate-induced Oxidative Toxicity inHippocampus. Glutamate is the major excitatory transmitter in the brain,and is proposed to play a role in epileptic pathogenesis and seizureactivity. Numerous in vivo models involving different kinds of seizuresand behavioral effects that are relevant for clinically distinct formsof epilepsy are known. In vitro models of glutamate-induced oxidativetoxicity are also known, an exemplary procedure described herein. Themouse hippocampal cell line (Davis & Maher (1994) Brain Res652(1):169-173) is maintained in Dulbecco's modified Eagles' medium(GibcoBRL of Gaithersburg, Md.) with 10% fetal bovine serum (AtlantaBiologicals of Atlanta, Ga.). HT22 cells are seeded onto 96-well platesat 20,000 cells per well and cultured overnight at 37° C. in normalgrowth medium. Glutamate-induced oxidative toxicity is elicited byadministration of 2-10 mM glutamate or NMDA. Further methods aredisclosed in Su et al. (1998) J Mol Cell Cardiol 30(3):587-598; Xiao etal. (1999) J Neurochem 72:95-101, and U.S. Pat. No. 6,017,965.

[0302] M.3. Assays for Recovery Following Ischemia or Other StressConditions

[0303] The effects of therapeutic compositions disclosed herein, may beexamined to determine potential therapeutic strategies for mitigatingand/or reversing cellular damage in these animal models. Exemplary,although not limiting, measures to assess therapeutic efficacy asdisclosed herein below.

[0304] Neurological Assessment Assay. Neurological deficit and recoverycan be monitored using standardized scores that represent carefulobservation of consciousness, respiration, cranial nerve activity,spinal nerve activity, and motor function, as disclosed in U.S. Pat. No.5,571,840. Interobserver variability can be resolved by consultation ofthe detailed description of each neurological function. Additionalassays of cognitive, sensory, and motor impairment are disclosed in U.S.Pat. No. 6,017,965.

[0305] Infarct Size Assay. The efficacy of candidate compounds disclosedherein can also be evaluated by determination of infarct size followingadministration of the composition to an animal subjected to ischemicconditions. At a selected timepoint(s) following initiation of ischemicconditions, such an animal is sacrificed and processed for routinehistology suitable for the tissue of interest and according to methodswell-known in the art Image processing software (e.g. Bio Scan OPTIMASof Edmonds, Wash.) can be utilized to facilitate accurate calculation ofinfarct volume.

[0306] Detection of Molecular Markers for Cell Degeneration. In anotherembodiment, damaged tissue can be identified in brain sections byimmunolabeling with antibodies that recognize antigens such as Alz-50,tau, A2B5, neurofilaments, neuron-specific enolase, and others that arecharacteristic of neurodegeneration as disclosed in U.S. Pat. No.6,046,381. Immunolabeled cells can be quantified using computer-aidedsemiquantitative analysis of confocal images.

[0307] Cell Viability Assay. When in vitro models of ischemia areemployed, cell viability can be assessed by measuring cell ability tometabolize 3-(4,5-dimethyidiazol-2-yl)-2,5-dipehnyltetrazolium bromide(MTT) as described in Hansen et al. (1989) Electrophoresis 10:645-652.Briefly 10 μl of MUT solution (5 mg/ml) is added to cell cultures is96-well plates and the cells are maintained in normal growth medium for4 hours at 37° C. Solubilization solution (100 μl; 50% dimethylformamideand 20% sodium dodecyl sulfate, pH 4.8) is then added directly to eachculture in individual wells of the 96-well plate. After an overnightincubation at room temperature, absorbance is measured.

[0308] Alternatively, cell viability can be assessed by measuring therelease of lactate dehydrogenase, a cytoplasmic enzyme that is releasedfrom dying cells as disclosed in Choi et al. (1987) J Neurosci 7:357 andU.S. Pat. No. 6,017,965.

[0309] Neuronal Growth Assays. A cell culture model of neural ischemiaas described herein above can be evaluated by visual examination oflabeled neuronal processes, and quantitation of the length, density, anddynamicism of neuronal processes (e.g. dendrites and spines) asdisclosed in Horch et al. (1999) Neuron 23:353-364 and McAllister et al.(1997) Neuron 18:767-778.

[0310] In another embodiment, molecular markers can be used to evaluateneurite growth in fixed brain tissue section. For example, brainsections derived from an animal model of ischemia can labeled usingantibodies that recognize MAP-2 (a marker of neuronal cell bodies anddendrites) and for synaptophysin (a marker of presynaptic terminals).Labeled sections can be viewed on a confocal microscope and documentedusing computer-aided semiquantitative analysis of confocal images. Thearea of the neuropil occupied by MAP-2-immunolabeled dendrites or bysynaptophysin-immunolabeled terminals can be quantified and expressed asa percentage of the total image area. See Masliah et al. (1992) ExpNeurol 136:107-122 and Toggas et al. (1994) Nature 367:188-193.

[0311] Additional methods for assaying neuronal growth are disclosed inDoherty et al. (1995) Neuron 14:57-66, Schnell et al. (1990) Nature343:269-272, U.S. Pat. Nos. 5,250,414 and 5,898,066, and InternationalPCT Publication WO 99/61585.

[0312] N. Disorders of Protein Transport

[0313] Protein misfolding disorders are a common component of numerousgenetic disease states including, but not limited to, cystic fibrosis,familial hypercholesterolemia, retinitis pigmentosa and α1-antitrypsinmisfolding. Compounds that modulate the activity of the Hsp90 family ofmolecular chaperones can thus be used in accordance with a therapeuticmethod of the present invention for reversing the protein foldingdefects that identify the disease state or for enhancing proteintransport from the endoplasmic reticulum of a cell. Thus, a compoundthat modulates the conformation of GRP94 can be used to treat a diseasestate resulting from defects in protein transport into or from theendoplasmic reticulum. Compounds that abrogate GRP94 activity can beused for the treatment of a disease state, such as cancer, wherein atherapeutic benefit can be provided by blocking the egress of proteins(e.g., growth factors) from the endoplasmic reticulum. conversely,compounds that promote GRP94 activity can be used to treat a diseasewherein a therapeutic benefit can be provided by enhancing proteinexport from the endoplasmic reticulum.

[0314] To assess misregulation of protein transport, a model system thatmeasures epidermal growth factor receptor (EGF-R) levels and/orintracellular localization can be employed (Supino-Rosin et al. (2000) JBiol Chem 275(29):21850-21855). For example, the benzoquinone ansamaycingeldanamycin targets two Hsp90 molecular chaperones (Hsp90 and GRP94)and by inhibiting their activities, blocks and promotes its subsequentproteolytic degradation. In response to geldanamycin treatment, EGF-R isunable to traffic to the plasma membrane and the cell becomes refractoryto stimulation by EGF.

EXAMPLES

[0315] The following Examples have been included to illustrate preferredmodes of the invention. Certain aspects of the following Examples aredescribed in terms of techniques and procedures found or contemplated bythe present inventors to work well in the practice of the invention.These Examples are exemplified through the use of standard laboratorypractices of the inventors. In light of the present disclosure and thegeneral level of skill in the art, those of skill will appreciate thatthe following Examples are intended to be exemplary only and thatnumerous changes, modifications and alterations can be employed withoutdeparting from the spirit and scope of the invention.

Examples 1-8 Ligand-Mediated Activation of GRP94 Molecular ChaperoneActivity

[0316] The amino terminal domain of Hsp90 chaperones contains anadenosine nucleotide binding pocket that binds the Hsp90 inhibitorsgeldanamycin and radicicol. The following Examples 1-8 demonstrate thatbis-ANS (1-1′ bis(4-anilino-5-napthalenesulfonic acid)), anenvironment-sensitive fluorophore that interacts with nucleotide bindingsites, binds to the adenosine nucleotide binding domain of GRP94 andactivates its peptide binding and molecular chaperone activities.Bis-ANS, like heat shock, elicits a tertiary conformational change inGRP94 which activates GRP94 function and is inhibited by radicicol.Confirmation of the N-terminal nucleotide-binding domain as the bis-ANSbinding site was obtained by sequencing of bis-ANS-labeled GRP94protease digestion products. These data identify a ligand-dependent,allosteric regulation of GRP94 and suggest a model for ligand-mediatedregulation of GRP94 function.

Materials and Methods for Examples 1-8

[0317] Materials. Fluorescent probes were obtained from Molecular Probes(Eugene, Oreg.). Bis-ANS concentration was determined by absorbance at385 nm (ε₃₈₅=16,790 cm⁻¹ M⁻¹ in water). Citrate synthase (E.C. 4.1.3.7)was purchased from Boehringer Mannheim (Mannheim, Germany). Radicicolwas obtained from Dr. Len Neckers, National Cancer Institute, Frederick,Md. Peptide VSV8 (RGYVYQGL—SEQ ID NO: 1) was synthesized by theUniversity of North Carolina at Chapel Hill Peptide Synthesis Facility(Chapel Hill, N.C.). Na [¹²⁵I] was purchased from Amersham Pharmacia(Piscataway, N.J.). All other reagents were obtained from Sigma ChemicalCo. (St. Louis, Mo.) unless otherwise indicated. GRP94 was purified fromporcine pancreas as described by Wearsch & Nicchitta (1996b)Biochemistry 35:16760-16769. The concentration of GRP94 was determinedby absorbance at 280 nm (1 mg/ml=1.18A₂₈₀).

[0318] Fluorophore Binding Reactions. All binding reactions, with theexception of the indicated circular dichroism and citrate synthaseaggregation experiments, were conducted in buffer A (110 mM KOAc, 20 mMNaCl, 2 mM Mg(OAc)₂, 25 mM K-HEPES pH 7.2, 100 μM CaCl₂). Fluorescentprobe and radicicol stocks were prepared in dimethyl formamide at 5 mMfinal concentration. For all assays, control reactions at solventdilutions identical to experimental conditions were performed to correctfor any solvent effects. Where indicated, GRP94 was heat shocked byincubation in a 50° C. water bath for 15 minutes followed by cooling to37° C.

[0319] Fluorescence Measurements. Emission spectra were obtained in aFLUOROMAX™ spectrofluorometer (SPEX Industries Inc. of Edison, N.J.)operating in photon counting mode. Spectra were recorded and processedwith DM3000 f operating software, version 2.1 (SPEX Industries Inc. ofEdison, N.J.). For emission scans, slit width was set at 1 nm.Excitation wavelengths were as follows: Prodan, 360 nm; ANS, 372 nm;bis-ANS, 393 nm; tryptophan, 295 nm. All spectra were backgroundcorrected.

[0320] Circular Dichroism Measurements. Far-UV CD spectrometry wasperformed on an AVIV Associates 62DS™ circular dichroism spectrometer(AVIV Associates of Lakewood, N.J.). Samples were analyzed in a 1 mmpath length quartz cuvette at 37° C. GRP94 samples (1 μM) were preparedin standard phosphate buffered saline solution as buffer A producedunacceptable dynode voltages in the relevant region of the spectrum.GRP94 was incubated with 10 μM bis-ANS for 2 hours at 37° C. prior toobtaining spectra. Spectra were recorded from 300 to 195 nm. Theα-helical content of GRP94 was calculated from the molar ellipticity at222 nm. See Myers & Jakoby (1975) J Biol Chem 250:3785-3789.

[0321] Conformational Analysis by Proteolysis. The conformational stateof GRP94 was assessed by tryptic digestion of the protein and subsequentSDS-PAGE analysis. For simple proteolysis experiments, 10 μl of a 0.5mg/ml GRP94 stock, with or without prior heat shock, was combined with 1μl bis-ANS and/or radicicol stock solutions and incubated for theindicated times at 37° C. Samples were then combined with 0.1% trypsinand digested for 30 minutes at 37° C. An equal volume of SDS-PAGE samplebuffer was added and the samples were snap frozen in liquid nitrogen.Immediately prior to gel analysis, samples were thawed and boiled for 5minutes. Samples were then separated on 12.5% SDS-polyacrylamide gels.Gels were fixed and stained with Coomassie Blue. For time courseexperiments, excess free bis-ANS was removed immediately prior totrypsinization by gel filtration on 0.5 ml G-25 SEPHADEX® spin columns.

[0322] Identification of the bis-ANS binding site. The bis-ANS bindingregion of GRP94 was identified by covalent incorporation of bis-ANS intoGRP94 following bis-ANS photolysis procedures described by Sharma et al.(1998) J Biol Chem 273(25):15474-78 and Seale et al. (1998) MethodsEnzymol 290:318-323. Briefly, 50 μg of GRP94 was combined with 50 μMbis-ANS in a final volume of 100 μl and photo-crosslinked for 15 minuteson ice with a 366 nm hand-held UV lamp (Ultra-violet Products, Inc. ofSan Gabriel, Calif.). Following photocrosslinking, GRP94-bis-ANScomplexes were digested with trypsin for one hour at 37° C. Thetrypsin-derived limit digestion products were then separated by C-18reverse phase HPLC using a continuous acetonitrile/water gradient in 20mM ammonium bicarbonate, with sequential detection by UV absorbance (220nm) and fluorescence emission (excitation 418 nm; emission 498 nm). Themajor resultant fluorescent peak was collected and the correspondingpeptide sequenced by Edman degradation on an Applied Biosystems PROCISE™model 492 automated protein sequencer.

[0323] Native Blue Electrophoresis. The oligomeric state of GRP94 wasassayed by blue native polyacrylamide gel electrophoresis (BN-PAGE) asdescribed by Schagger et al. (1994) Anal Biochem 217:220-230. GRP94 waseither heat shocked or exposed to a 10-fold molar excess of bis-ANS forthe indicated times. Samples were then dissolved in 15% glycerol andloaded onto 5-18% gradient gels with 0.02% Coomassie Brilliant Blue inthe cathode buffer. Gels were run at 4° C., stained with Coomassie Blue,destained and dried.

[0324] Citrate Synthase Aggregation Assays. The effects of GRP94 on thethermal aggregation of citrate synthase were assayed by the methodsdescribed by Buchner et al. (1998) Methods Enzymol 290:323-338. Samplescontaining no protein, or GRP94 (1 μM), were incubated in 40 mM HEPES pH7.5 for two hours at 37° C. with either 0.2% DMF or 10 μM bis-ANS. Thesamples were then warmed to 43° C. for five minutes and placed in aspectrofluorometer thermostatted at 43° C. Citrate synthase was thenadded to 0.15 μM final concentration and the thermal aggregation of theenzyme followed by light scattering. Excitation and emission wavelengthswere both 500 nm with 2 nm slit width. The time course of citratesynthase aggregation was followed for 1000 seconds.

[0325] Peptide Binding to GRP94. Iodination of VSV8 was performed by theIODOBEADS™ procedure (Pierce Chemical Co. of Chicago, Ill.), andunincorporated [¹²⁵I] was removed by fractionation on a SEP-PAK™ C18reverse-phase cartridge. lodinated peptide was mixed with unlabeledpeptide to yield a final specific activity of 6.0 μCi/mg. GRP94 (4.7 μg,final concentration 0.5 μM) was incubated with an equimolar quantity ofbis-ANS in 0.1% DMF in 100 μL buffer A for 3.5 hr at 37° C. Samples werethen incubated for an additional 30 min at 37° C., or heat shocked for15 min at 50° C. and allowed to recover for 15 min at 37° C. A ten-foldmolar excess of [¹²⁵I ]VSV8 was added (final concentration 5 μM) and themixture incubated for 30 min at 37° C. All incubations were performed inthe dark to prevent bis-ANS degradation. Samples were then eluted on1.2-mL SEPHADEX® G-75 spin columns pre-blocked with 75 μg BSA, and[¹²⁵I] was quantitated by gamma counting.

Example 1 Binding of Polarity-Sensitive Fluorescent Probes to GRP94

[0326] Recent studies on the conformational regulation of GRP94 haveidentified a tertiary structural change that occurs in response to heatshock and is associated with an activation of peptide binding activity.See Wearsch et al. (1998) Biochemistry 37(16):5709-16, Sastry &Linderoth (1999) J Biol Chem 274:12023-12035. Coincident with the heatshock-elicited conformational change, GRP94 displays enhanced binding ofenvironment sensitive fluorescent probes such as Nile Red, whichpreferentially bind to hydrophobic domains (Wearsch et al., 1998). GRP94contains two domains of significant hydrophobicity, a C-terminalassembly domain and a highly conserved N-terminal region, whichcorresponds to the Hsp90 geldanamycin and adenosine nucleotide bindingsite. See Stebbins et al. (1997) Cell 89:239-250; and Prodromou et al.(1997) Cell 90:65-75.

[0327] To characterize the structural basis for the heat shock dependentactivation of GRP94 activity, the interaction of polarity-sensitivefluorophores with native and heat shocked GRP94 was examined. The threeprobes tested, Prodan (6-propionyl-2-(dimethylamino)naphthalene), 8-ANS(1,8-anilinonaphthalenesulfonate) and bis-ANS(bis(1,8-anilino-naphthalenesulfonate) are structurally related probesthat bind to hydrophobic sites on proteins and undergo substantialfluorescence spectrum changes upon introduction into nonpolarenvironments, as discussed by Rosen & Weber (1969) Biochemistry8:3915-3920; Weber & Farris (1979) Biochemistry 18:3075-3078; Takashi etal. (1977) Proc Natl Acad Sci USA 74:2334-2338; Shi et al. (1994)Biochemistry 33:7536-7546. The following experimental protocol wasutilized. GRP94 was warmed to 37° C. and either maintained at 37° C. orheat shocked for 15 minutes at 50° C., followed by incubation at 37° C.Subsequently, probe stocks were added to the GRP94 stocks and emissionspectra recorded after 30 min at 37° C.

[0328] As depicted in FIG. 1A, the emission maxima of Prodan in thepresence of native or heat shocked GRP94 were essentially identical,indicating that Prodan does not interact with the hydrophobic bindingpocket(s) displayed by heat shocked GRP94. In contrast, the structurallyrelated probe, 8-ANS, displays weak interactions with native GRP94, yetbinds avidly following heat shock (FIG. 1B).

[0329] The interaction of bis-ANS with GRP94 was complex, and displayeda clear time dependence. As depicted in FIGS. 1C and 1D, the initialbis-ANS binding to native GRP94 was bi-phasic and following extendedincubations in the presence of bis-ANS, a level of fluorophore bindingsimilar to that seen with heat shocked GRP94 was observed. These datasuggest that maximal bis-ANS binding to GRP94 required a slow structuraltransition. This transition further suggests a bis-ANS elicitedconformational change in GRP94 and/or the bis-ANS dependentstabilization of a conformation state accessed at low frequency by thenative protein.

Example 2 Analysis of bis-ANS Binding to Heat Shocked GRP94

[0330] To determine the affinity of bis-ANS for GRP94, bis-ANS was addedto increasing concentrations of heat shocked GRP94, the fluorescencespectrum was determined, and the emission intensity at 475 nm plotted asa function of GRP94 concentration (FIGS. 2A and 2B). Under theexperimental conditions used in this series of experiments, bis-ANSbinding to GRP94 was near maximal at a 20-fold molar excess of GRP94monomer over bis-ANS, with half maximal binding observed at 110 nM GRP94(FIG. 2B). Importantly, these data indicate that bis-ANS binds in asaturable manner to heat shocked GRP94 and that the site(s) of bis-ANSbinding to GRP94 displayed similar relative affinities for bis-ANS.

Example 3 Structural Consequences of bis-ANS Binding to GRP94

[0331] Following an extended incubation period, the emission spectra ofbis-ANS bound to native GRP94 bears substantial similarity to thatemission spectra of bis-ANS bound to heat shocked GRP94. Because heatshock is known to elicit a stable tertiary conformational change inGRP94 (Wearsch et al. (1998) Biochemistry 37(16):5709-16) these datasuggest that the binding of bis-ANS to GRP94 induces, or stabilizes, aconformational change similar to that occurring in response to heatshock. To determine whether the GRP94 conformation seen upon addition ofbis-ANS is similar to that observed following heat shock, a series ofstructural studies on the bis-ANS/GRP94 complex was performed.

[0332] In one series of experiments, the proteolysis patterns of native,heat shocked and bis-ANS treated GRP94 were examined. As shown in FIG.3A, lanes 2 and 3, incubation of native GRP94 with low levels of trypsinyields two prominent proteolysis products, representing known structuraldomains of the protein, as described by Stebbins et al. (1997);Prodromou et al. (1997) Cell 90:65-75; Wearsch & Nicchitta (1996b)Biochemistry 35:16760-16769. In contrast, proteolysis of either bis-ANStreated or heat shocked GRP94 yields a substantially reduced recovery ofthe prominent proteolysis products, with the concomitant appearance of adiverse array of proteolytic fragments of higher SDS-PAGE mobility.Essentially identical proteolysis patterns were observed followingeither heat shock or bis-ANS treatment of HSP90.

[0333] These data provide evidence that bis-ANS binding to GRP94 elicitsor stabilizes GRP94 in a conformation similar to that occurring inresponse to heat shock, suggesting that there exists a GRP94conformation state that can be readily accessed and/or stabilized byeither heat shock or ligand (bis-ANS) binding.

Example 4 Effects of bis-ANS Binding on GRP94 Quaternary and SecondaryStructure

[0334] When purified from tissue, GRP94 exists as a homodimer, asdescribed by Wearsch & Nicchitta (1996a) Prot Express Purif 7(1):114-21;Nemoto et al. (1996) J Biochem 120:249-256. Following heat shockhowever, GRP94 forms higher molecular weight complexes, as described byWearsch et al. (1998) Biochemistry 37:5709-5719. To further characterizethe effects of bis-ANS on GRP94 structure, the oligomerization states ofnative, heat shocked and bis-ANS treated GRP94 were assayed by the bluenative polyacrylamide gel electrophoresis (BN-PAGE) technique describedby Schagger et al. (1994). In these experiments, GRP94 was incubatedwith bis-ANS or briefly heat shocked and subsequently incubated at 37°C. The samples were then analyzed by BN-PAGE. As seen in FIG. 4, in theabsence of heat shock or bis-ANS treatment the majority of GRP94 existsas a dimer with an apparent molecular weight of approximately 200 kDa.However, exposure to heat shock causes a relatively rapid formation oftetramers, hexamers, and octamers (FIG. 4, lanes 2-4). Incubation ofGRP94 with a ten-fold molar excess of bis-ANS induces changes in thequaternary structure of GRP94 that mimic those seen upon heat shock(FIG. 4, lanes 4, 5). These data lend further support to the hypothesisthat bis-ANS induces or stabilizes a structural transition in GRP94 thatis similar to that occurring in response to heat shock.

[0335] To gain further insight into the nature of the bis-ANS dependentconformational change, GRP94 was subjected to heat shocked or treatedwith bis-ANS and far-UV CD spectra obtained (FIG. 5). As shown in FIG.5, the CD spectra for native, heat shocked, and bis-ANS treated GRP94were identical, indicating that bis-ANS binding does not alter GRP94secondary structure.

Example 5 Radicicol Inhibits Temperature and bis-ANS Induced GRP94Conformational Changes

[0336] Radicicol, a macrocyclic antibiotic, binds to the highlyconserved N-terminal nucleotide binding pocket of HSP90 and therebyblocks HSP90 function. (Sharma et al. (1998) Oncogene 16(20):2639-45;Roe et al. (1999) J Med Chem 42:260-266). To determine if radicicolbinding also influenced the structural dynamics of GRP94, the followingexperiments were performed. GRP94 was incubated with increasingconcentrations of radicicol, heat shocked, cooled, and digested withtrypsin. Subsequent SDS-PAGE analysis of the samples showed that in thepresence of radicicol, GRP94 was unable to undergo the heatshock-induced structural transition, as assayed by the similarities inproteolysis patterns between native GRP94 and radicicol-treated, heatshocked GRP94. Similar inhibition of the heat shock induced structuraltransition of HSP90 by radicicol was also observed.

[0337] To determine if radicicol could also inhibit the bis-ANSdependent GRP94 structural transition, GRP94 was incubated withincreasing concentrations of radicicol, bis-ANS was then added, and thesamples were incubated for one hour. Samples were subsequently digestedwith trypsin and the proteolysis patterns determined by SDS-PAGE. As isdepicted in FIG. 6A, radicicol, when present at a ten-fold molar excessover bis-ANS, efficiently blocked the bis-ANS-dependent GRP94conformation change.

[0338] Though the experiment depicted in FIG. 6A indicated thatradicicol was able to inhibit the appearance of the bis-ANS-dependentconformational state, it was necessary to determine if bis-ANS bindingto GRP94 was blocked by radicicol treatment. To this end, the followingexperiment was performed. GRP94 was incubated in the presence ofincreasing concentrations of radicicol, subsequently heat treated underconditions sufficient to elicit efficient bis-ANS binding, and bis-ANSbinding assayed. As shown in FIG. 6B, radicicol, in a dose-dependentmanner, inhibited bis-ANS binding to heat-treated GRP94.

[0339] Because radicicol itself blocks the heat shock-inducedconformation change, these data present two models of bis-ANS action. Inone model, bis-ANS binds to the nucleotide binding domain and directlyelicits the observed conformational change. Radicicol, by binding to theadenosine nucleotide binding pocket, would then be predicted to inhibitthe bis-ANS-dependent conformational change. In an alternative model,GRP94 interconverts, in a temperature sensitive manner, between twoconformational states, arbitrarily referred to as the open or the closedstate. In the open state, bis-ANS bind and thereby stabilizes the openconformation whereas radicicol binding would stabilize the closedconformation. For both models, bis-ANS binding to the N-terminaladenosine nucleotide binding domain was predicted and was subsequentlyexamined.

Example 6 bis-ANS binds to the N-terminal AdenosineNucleotide/Radicicol/Geldanamycin Binding Domain

[0340] Having determined that bis-ANS can alter the conformation ofGRP94, the site of bis-ANS binding to GRP94 was targeted foridentification. Irradiation of bis-ANS with UV light allows the covalentincorporation of the probe into protein binding sites, as described bySharma et al. (1998) J Biol Chem 273(25):15474-78 and Seale et al.(1998) Methods Enzymol290:318-323. As described in Materials andMethods, GRP94 was combined with an excess of bis-ANS andphoto-crosslinked on ice for 15 minutes. GRP94 was subsequently digestedwith trypsin, the fluorescent peptides purified by HPLC, and thesequence of the labeled peptides determined by Edman sequencing. Themajor resultant fluorescent peptide yielded the sequence YSQFINFPIYV(SEQ ID NO: 2), which mapped to residues 271-281 of the N-terminaldomain of GRP94. This segment is homologous to the human HSP90 sequenceHSQFIGYPITLFV (SEQ ID NO: 3) from amino acids 210-222, and overlaps withthe C-terminal region of the adenosine nucleotide/geldanamycin/radicicolbinding domain (Stebbins et al. (1997) Cell 89:239-250; Prodromou et al.(1997) Cell 90:65-75).

Example 7 Bis-ANS Activates GRP94 Chaperone Activity

[0341] To determine if the bis-ANS-dependent conformational changes inGRP94 were of functional significance, the molecular chaperoneactivities of native, heat shocked and bis-ANS treated GRP94 wereevaluated in a thermal aggregation assay, as described by Jakob et al.(1995) J Biol Chem 270:7288-7294 and Buchner et al. (1998) MethodsEnzymol 290:323-338. In these experiments, citrate synthase aggregationwas assayed in the presence of buffer, native GRP94, heat shocked GRP94or GRP94 that had been previously exposed to bis-ANS for two hours.Following experimental treatment of the GRP94, reactions wereequilibrated at 43° C., citrate synthase then added and aggregation, asrepresented by light scattering, was measured.

[0342] In the absence of GRP94, citrate synthase undergoes rapid thermalaggregation and under the experimental conditions depicted in FIG. 7A,reaches a plateau level within 15 min. In the presence of native GRP94,the degree of aggregation is reduced, suggesting that at least afraction of the population of native GRP94 molecules are in an activeconformation. Under these experimental conditions, approximately 50% ofthe citrate synthase aggregated. At the concentration of GRP94 used inthese experiments, and assuming a stoichiometric interaction, theseresults indicate that roughly 8% of the native GRP94 is in the activeconformation. In the presence of heat shocked or bis-ANS treated GRP94,no thermal aggregation of citrate synthase was detectable (FIG. 7A).These data indicate that the ability of GRP94 to bind to substrateproteins is enhanced by prior heat shock or bis-ANS treatment andsuggest that the GRP94 conformation elicited by heat shock or bis-ANSbinding represents an active state of the molecule.

Example 8 bis-ANS Activates Peptide Binding Activity to GRP94

[0343] To assess the effects of bis-ANS treatment on the peptide bindingactivity of GRP94, GRP94 was either treated with bis-ANS, or brieflyheat shocked. A ten-fold molar excess of [¹²⁵I]-VSV8 was then added andthe mixture incubated for 30 min at 37° C. Free peptide was separatedfrom bound peptide by SEPHADEX® 75 spin column chromatography and thebound peptide was quantitated by gamma counting. As shown in FIG. 7B,treatment of GRP94 with bis-ANS significantly enhanced the peptidebinding activity of GRP94, yielding approximately a four to five-foldstimulation over native protein. Under similar conditions, heat shockedGRP94 displayed approximately a ten-fold stimulation of binding. Fromthe data presented in FIGS. 7A and 7B, it is apparent that bis-ANSelicits or stabilizes a GRP94 conformation that displays markedlyenhanced molecular chaperone and peptide binding activities.

Summary of Examples 1-8

[0344] Examples 1-8 demonstrate that bis-ANS binds to the conserved,N-terminal adenosine nucleotide binding domain of GRP94 and elicits atertiary conformational change yielding markedly enhanced molecularchaperone and peptide binding activities. The binding of bis-ANS toGRP94 is bi-phasic, with an initial rapid binding phase followed by aslow, extended binding phase. In accord with these data, bis-ANS bindsto and stabilizes a low abundance GRP94 conformation, referred to as theopen state. In this model, GRP94 molecular chaperone and peptide bindingactivity is intimately coupled to such a conformation change. While itis not applicants' desire to be bound by any particularly theory or act,in the absence of regulatory ligands, access to this conformation isbelieved to occur in a time and temperature-dependent manner throughintrinsic structural fluctuations. Inhibitory ligands, such asgeldanamycin and radicicol, function by binding to and stabilizing GRP94in a closed, or inactive, conformation.

[0345] Summarily, Examples 1-8 disclose the identification of a ligandelicited conformational change in GRP94 that is accompanied by a markedactivation of molecular chaperone and peptide binding activities. Thesimilarities between the conformations of GRP94 following heat shockactivation and bis-ANS binding support the conclusion that GRP94conformation and activity can be regulated by ligand binding to theN-terminal adenosine nucleotide binding domain and that the conformationof the protein in the bis-ANS liganded state is physiologicallyrelevant.

Examples 9-13 Allosteric Ligand Interactions in the Adenosine NucleotideBinding Domain of the Hsp90 Chaperone, GRP94

[0346] Examples 9-13 disclose that GRP94 and HSP90 differ in theirinteractions with adenosine-based ligands. GRP94 displayed high affinitysaturable binding of the adenosine derivativeN-ethylcarboxamido-adenosine (NECA), whereas HSP90 did not. In NECAdisplacement assays, GRP94 exhibited weak binding affinities for ATP,ADP, AMP, adenosine and cAMP. GRP94 ATPase activity, though present, wasnon-saturable with respect to ATP concentration and thus could not becharacterized by traditional enzymatic criteria. Radioligand andcalorimetric studies of NECA binding to GRP94 revealed that ligandbinding to the nucleotide binding domain is under allosteric regulation.GRP94 is thus regulated through a ligand-based allosteric mechanism andthrough regulatory adenosine-based ligand(s) other than ATP.

Materials and Methods for Examples 9 -13

[0347] Purification of GRP94, BiP and Hsp90. GRP94 was purified fromporcine pancreas rough microsomes as described by Wearsch & Nicchitta(1996a) Prot Express Purif 7:114-121 with the following modifications.Rough microsomes were washed after the initial isolation by 10-folddilution in 0.25M sucrose, 20 mM KOAc, 25 mM K-Hepes, pH 7.2, 5 mMMg(OAc)₂ and subsequent re-isolation by centrifugation (30 min, 40K rpm,4° C., Ti50.2 rotor). To release the lumenal contents from the isolatedrough microsomes, the microsomes were permeabilized by addition of 5 mMCHAPS and the lumenal contents were subsequently isolated bycentrifugation for 2 hours at 45,000 RPM (4° C., Ti50.2 rotor).

[0348] BiP was purified by the following procedure. A lumenal proteinfraction obtained from porcine pancreas rough microsomes was cycledovernight through a 1 ml ADP-agarose and a 1 ml ATP-agarose (SigmaChemical Co. of St. Louis, Mo.) column coupled in series. The columnswere then washed with 2×5 ml of a buffer containing 350 mM NaCl, 25 mMTris, pH 7.8, 5 mM Mg² ⁺ and the BiP was eluted from the nucleotideaffinity columns with 3×5 ml of the identical buffer supplemented with10 mM ATP and ADP. The BiP containing fractions were identified bySDS-PAGE, and dialyzed against 2×4 L of buffer A (110 mM KOAc, 20 mMNaCl, 25 mM K-Hepes, pH 7.2, 2 mM Mg(OAc)₂ 0.1 mM CaCl₂). The proteinsample was then applied to a SUPERDEX® 26/60 column (Amersham PharmaciaBiotech of Piscataway, N.J.) equilibrated in buffer A, and the BiPcontaining fractions, again identified by SDS-PAGE, were pooled andconcentrated by centrifugal ultrafiltration (CENTRICON-30®; Amicon ofBeverly, Mass.).

[0349] Hsp90 was purified from rat liver cytosol as follows. Cytosol wasadjusted to 30% ammonium sulfate and stirred for 60 min on ice. Thesolution was centrifuged at 20,000×g in a Sorvall SS34 rotor for 15minutes and the supernatant collected and filtered through a 0.22 μmfilter. The filtered supernatant was supplemented with proteaseinhibitors (1 μg/ml pepstatin, 1 μg/ml leupeptin, 20 μg/mi SBTI, and 0.5mM PMSF) and loaded onto a phenyl-SUPEROSE™ HR10/10 column (AmershamPharmacia Biotech of Piscataway, N.J.). After washing, the boundproteins were eluted with a gradient of 30-0% saturated ammonium sulfatein 10 mM Tris/HCl, pH 7.5, 1mM EGTA, 0.5 mM DTT and the Hsp90 containingfractions were identified by SDS-PAGE. The Hsp90 containing fractionswere then pooled and dialyzed 2×3 hr against 2 L of low salt buffer (10mM NaCl, 25 mM Tris, pH 7.8). The dialyzed sample was then filteredthrough a 0.22 μm filter, and injected onto a MONO-Q™ HR 10/10 column(Amersham Pharmacia Biotech of Piscataway, N.J.) equilibrated in lowsalt buffer. The column was eluted with a gradient of 10 mM-750 mM NaClin 25 mM Tris, pH 7.8. The Hsp90-containing fractions were identified bySDS-PAGE and pooled.

[0350] Further purification was achieved by applying the MONO-Q™ pool toa 4 mL hydroxylapatite column (Bio-Rad HTP of Hercules, Calif.)equilibrated in buffer B (10 mM NaH₂PO₄, pH 6.8, 10 mM KCl and 90 mMNaCl). The hydroxylapatite column was eluted with a 10 mM NaH₂PO₄ to 250mM NaH₂PO₄, gradient and the Hsp90 fractions were identified bySDS-PAGE. The Hsp90 pool, in 225 mM NaH₂PO₄, 10 mM KCl, and 90 mM NaCl,was concentrated by centrifugal ultrafiltration (CENTRICON®-30; Amicon,Beverly, Mass.) and stored at −80° C.

[0351] [³H]-NECA Binding Assay. Nine pg of GRP94 was incubated with 20nM [³H]-NECA (Amersham Pharmacia Biotech of Piscataway, N.J.), andvarious concentrations of competitors for one hour on ice in a finalvolume of 250 μl of 50 mM Tris, pH 7.5. Where indicated, bindingreactions were performed in either buffer C (10 mM Tris, pH 7.5, 50 mMKCl, 5 mM MgCl₂, 2 mM DTT, 0.01% NP-40, 20 mM Na₂MoO₄) or 50 mM Tris, pH7.5, 10 mM Mg(OAc)₂. Bound versus free [³H]-NECA was assayed by vacuumfiltration of the binding reactions on #32 glass fiber filters(Schleicher and Schuell of Keene, N.H.), pre-treated with 0.3%polyethyleneimine (Sigma Chemical Co. of St. Louis, Mo.). Vacuumfiltration was performed with an Amersham Pharmacia Biotech (Piscataway,N.J.) vacuum filtration manifold.

[0352] Filters were rapidly washed with 3×4 ml of ice cold 50 mM Tris,pH 7.5, placed in 5 ml of scintillation fluid (SAFETYSOLVE™, RPI of Mt.Prospect, Ill.), vortexed, and counted by liquid scintillationspectrometry. In experiments in which the kinetic parameters of[³H]-NECA binding to GRP94 were determined, the chemical concentrationand specific activity of NECA was adjusted by addition of unlabeledNECA. All binding reactions were performed in triplicate and correctedby subtraction of background values, determined in binding reactionslacking GRP94.

[0353] ATP Binding Assay. Six μg of GRP94, BiP, and Hsp90 was incubatedwith 50 μM yZ[³²P] ATP (1000 μCi/μmol) (Amersham Pharmacia Biotech ofPiscataway, N.J.) in buffer B on ice for 1 hour. Nitrocellulose filters(BA85) (Schleicher & Schuell of Keene, N.H.) were individually wet inbuffer B before use, and bound versus free [³²P]-ATP was separated byvacuum filtration. Filters were washed with 3×2 mL of ice cold buffer B,placed in 5 mL of scintillation fluid, vortexed, and counted.

[0354] Isothermal Titration Calorimetry. Isothermal calorimetryexperiments were performed at 25° C. using a MSC calorimeter (MicroCalInc. of Northampton, Mass.). To determine the NECA binding parameters,two 5 μl injections were followed by twenty-three 10 μL injections froma 152 μM NECA stock. The reaction chamber (1.3 mL) contained 5 μM GRP94.Necessary corrections were made by subtracting the heats of dilutionresulting from buffer addition to protein solution and ligand solutioninto buffer. The corrected data were then fit by the ORIGIN™ software(Microcal Software, 1998) to obtain the binding parameters. Theradicicol binding parameters were obtained in a similar manner with 5 μMGRP94 and 115 μM radicicol.

[0355] Phosphorylation Assays. To assay for GRP94 autophosphorylation, 1μM GRP94 was incubated with γ-[³²P]ATP (6000 cpm/pmol) (AmershamPharmacia Biotech of Piscataway, N.J.), diluted with cold ATP to yield afinal concentration of 0.15 mM ATP in a buffer containing 10 mM Mg(OAc)₂and 50 mM K-Hepes, pH 7.4. For the casein kinase assay, 1 unit of caseinkinase II was incubated as described above, with the addition of 4 μMcasein. Competitors were added to the appropriate samples to yield finalconcentrations of 180 μM NECA in 3.6% DMSO, 180 μM radicicol in 3.6%DMSO, 5 μg/ml heparin, 5 mM GTP, or 3.6% DMSO. The 25 μl reactionmixtures were incubated at 37° C. for 1 hour and quenched by addition of10% trichloroacetic acid. Samples were analyzed by 10% SDS-PAGE gels andthe phosphorylated species were quantitated using a Fuji MACBAS1000™phosphorimaging system (Fuji Medical Systems of Stamford, Conn.).

[0356] ATPase Assay. 100 μl reactions consisting of 1 μM GRP94 monomer,various concentrations of MgATP (pH 7.0), and 50 mM K-Hepes, pH 7.4,were incubated for two hours at 37° C. Samples were then spun through aCENTRICONO®30 (Amicon of Beverly, Mass.) at 10,000 rpm, 42° C. toseparate protein from nucleotide. A final concentration of 50 mM(NH₄)₂HPO₄, pH 7.0, and 4 μM AMP, pH 7.0, was added to dilutions of theabove samples and centrifuged at 15,200 rpm for 5 minutes at 4° C. 100μL of supernatant was then fractionated on a PARTISIL™ SAX column(Alitech of Deerfield, Ill.), using a Series 1050 Hewlett Packard HPLCsystem. Elution of nucleotides was performed by step gradient elutionusing a mobile phase of 150 mM (NH₄)₂HPO₄, pH 5.2, at 1.2 ml/min for thefirst ten minutes, followed by 300 mM (NH₄)₂HPO₄, pH 5.2, at a flow rateof 2 ml/min for the remainder of the elution. In this protocol, ADP andATP were well resolved, with ADP eluting at 7 minutes and ATP at 12minutes. Peak height values were used in calculations of percenthydrolysis and ADP formation. Spontaneous hydrolysis was determined foreach ATP concentration in paired incubations lacking GRP94. The AMP wasused as an internal reference standard to control for equivalent sampleloading.

[0357] Tryptophan Fluorescence. Tryptophan fluorescence measurementswere conducted in a FLUOROMAX™ spectrofluorometer (Spex Industries, Inc.of Edison, N.J.) with the slit widths set to 1 nm for both excitationand emission. Samples were excited at a wavelength of 295 nm and theemission spectra were recorded from 300-400 nm. All spectra werecorrected by subtraction of buffer or buffer plus ligand samples. GRP94(50 μg/mi) was incubated in buffer A supplemented with 10 mM Mg(OAc)₂and the following concentrations of ligands for 1 hour at 37° C. (50 μMNECA, 50 μM geldanamycin, 2.5 mM ATP, or 2.5 mM ADP). Samples were thencooled to room temperature, transferred to a quartz cuvette, and thespectra collected. In control experiments, free tryptophan fluorescencewas not significantly influenced by the presence of any of the assayedligands.

Example 9 Hsp90 Proteins Differ in Adenosine-based Ligand BindingProperties

[0358] To determine whether Hsp90 and GRP94 displayed distinctadenosine-ligand binding properties, the relative NECA and ATP bindingactivities of GRP94, Hsp90 and BiP, the endoplasmic reticulum Hsp70paralog, were compared (FIG. 8). In these assays, purified GRP94, Hsp90or BiP were incubated on ice for 60 min in the presence of 20 nM[³H]-NECA and the bound versus free NECA resolved by vacuum filtration.As is evident in FIG. 8, whereas GRP94 displayed readily detectable[³H]-NECA binding activity, [³H]-NECA binding was not observed for Hsp90or BiP. In similar experiments, [³H]-NECA binding to Hsp90 was evaluatedin the presence of molybdate and NP-40, which are known to stabilize theHsp90 conformation associated with ATP binding, as described by Sullivanet al. (1997). Under these conditions, [³H]-NECA binding to Hsp90 wasagain not observed.

[0359] When ATP binding was assayed, BiP displayed the expected ATPbinding activity whereas no ATP binding was observed to Hsp90 or GRP94.As discussed below, the inability to detect ATP binding to Hsp90 islikely a consequence of the low affinity of Hsp90 for ATP (Prodromou etal. (1997) Cell 90:65-75; Scheibel et al. (1997) J Biol Chem272:18608-18613). In summary, these data indicate that GRP94 and Hsp90differ in their ability to bind the adenosine-based ligand NECA, andsuggest that the ligand specificity of the adenosine nucleotide bindingpocket of GRP94 differs from that of Hsp90.

Example 10 Kinetic Analysis of NECA Binding to GRP94

[0360] A kinetic analysis of [³H]-NECA binding to mammalian GRP94 isdepicted in FIGS. 9A and 9B. [³H]-NECA binding to GRP94 was saturable,with a Kd of 200 nM and displayed a binding stoichiometry of 0.5 mol[³H]-NECA/mol GRP94 monomer. These values are similar to those observedwith placental GRP94 (adenotin) by Hutchison et al. (1990) Biochemistry29:5138-5144. A Hill plot of the binding data yielded a slope of 1.2,indicating that [³H]-NECA binding to GRP94 was not cooperative.

[0361] Structurally, GRP94 exists as a dimer of identical subunits asdescribed by Wearsch & Nicchitta (1996a) Prot Express Purif 7:114-121;Wearsch & Nicchitta (1996b) Biochemistry 35:16760-16769; Nemoto et al.(1996) J Biochem 120:249-256). Given that the two subunits areidentical, a 50% ligand occupancy at binding saturation was unexpected.The dissociation rate of NECA from GRP94 is rapid (Huttemann et al.(1984) Naunyn Schmiedebergs Arch Pharmacol. 325:226-33) and so it wasconsidered that the observed fractional occupancy level could reflect anartifact of the method used to separate bound vs. free [³H]-NECA.

[0362] To evaluate the accuracy of the half-site occupancy value, thekinetics of NECA-GRP94 interaction were evaluated by isothermaltitration calorimetry, a method that does not require the physicalseparation of bound and free ligand. In these experiments, illustratedin FIG. 9C, the binding stoichiometries of GRP94 for NECA and radicicolwere determined. Radicicol is an antibiotic inhibitor of Hsp90 functionthat binds to the N-terminal nucleotide binding pocket of Hsp90 withhigh affinity (19 nM) and the expected binding stoichiometry of 2 molradicicol/mol Hsp90 dimer, as proposed by Roe et al. (1999) J Med Chem42:260-266. Analysis of NECA binding to GRP94 by isothermal titrationcalorimetry yielded a binding stoichiometry of 1.1 mol NECA/mol GRP94dimer. (FIG. 9C).

[0363] Radicicol, in contrast, bound at a stoichiometry of 2 molradicicol/mol GRP94 dimer, as shown in FIG. 9C. These data indicate thatwhile radicicol can achieve full occupancy of the two nucleotide bindingsites present in the native GRP94 dimer, other ligands, such as NECA,either bind to a single unique site on GRP94, or upon binding to one ofthe nucleotide binding sites, elicit a conformational change in thepaired site that prevents further ligand binding.

Example 11 Specificity of Ligand Binding to the Nucleotide BindingPocket of GRP94

[0364] To determine whether NECA bound to a single unique site on GRP94or, alternatively, displayed half-site occupancy of the N-terminaladenosine nucleotide binding pockets, experiments were first performedto determine if NECA binds to the adenosine nucleotide binding pocket.[³H]-NECA competition assays were performed with geldanamycin andradicicol, both of which are known to bind with high affinities to thenucleotide binding pocket of Hsp90 (Roe et al. (1999) J Med Chem42:260-266, Lawson et al. (1998) J Cell Physiol 174:170-8). The datadepicted in FIG. 10A indicate that both geldanamycin and radicicolcompete with [³H]-NECA for binding to GRP94 and do so with high relativeaffinities and in the following rank order, radicicol >geldanamycin.

[0365] As described Wearsch & Nicchitta (1997) J Biol Chem272:5152-5156, it is difficult to detect stable binding of ATP to GRP94.Should GRP94 display a similar and quite low affinity for ATP, asreported for Hsp90 (Kd=132 μM) by Prodromou et al. (1997) Cell 90:65-75,it would be very unlikely that ATP binding could be detected by standardtechniques. Given the high affinity of GRP94 for NECA, however,potential interactions of NECA with the nucleotide binding domain couldbe addressed by competitive displacement assays. To determine thenucleotide binding specificity of GRP94, the ability of ATP, ADP or AMPto compete with NECA binding to GRP94 was examined. In theseexperiments, GRP94 was incubated with 20 nM [³H]-NECA in the presence ofincreasing concentrations of ATP, ADP or AMP and the relative [³H]-NECAbinding determined by vacuum filtration. In the presence of nominal (80μM) Mg²⁺, it was observed that ATP, ADP and AMP effectively competedwith [³H]-NECA for binding to GRP94.

[0366] Three points are evident from these experiments. One, becauseNECA binding to GRP94 can be effectively inhibited by geldanamycin,radicicol, and adenosine nucleotides, it can be concluded that NECAbinds to the analogous N-terminal adenosine nucleotide binding domain ofGRP94 (FIG. 10A). Two, the relative affinities of GRP94 for ATP, ADP andAMP are quite low (FIG. 10B). Thus, a 50% inhibition of [³H]-NECAbinding required approximately a 1000-fold molar excess of ATP. Three,the relatively high binding affinity of GRP94 for NECA, when viewed withrespect to the established molecular interactions of the adenine andribose moieties of adenosine in the adenosine nucleotide binding pocketof Hsp90, suggest that a principal selection for ligands is made on thebasis of the adenosine moiety. For this reason, the interaction of otheradenosine-bearing ligands with the N-terminal nucleotide binding pocketwas examined (FIG. 10C). These data indicated that cAMP and freeadenosine also bound to the N-terminal adenosine nucleotide bindingpocket of GRP94, with the relative displacement activity approximatingthat observed for ADP.

[0367] Because the data indicated that GRP94 bound adenosine, adenosinederivatives, and adenosine nucleotides with an unusually broadspecificity, additional studies were performed to confirm the nucleosidespecificity of these binding phenomena. In the experiment depicted inFIG. 11, the [³H]-NECA competitive displacement assay was used toaddress the nucleoside base specificity directly. Though GRP94 couldbind both ATP and deoxyATP, little to no binding of GTP, CTP or UTP wasobserved. The nucleotide binding pocket of GRP94 thus appears to bestrict in its selection of adenosine-bearing ligands.

[0368] In comparing the relative affinities of GRP94 for ATP and ADP, asdisplayed in NECA competition assays, clear differences between theATP/ADP binding properties of GRP94 and those previously reported forHsp90 were noted. Regarding GRP94, ATP was found to compete NECA bindingwith an eight-fold higher efficacy than ADP. In contrast, the N-terminaldomain of Hsp90 binds ADP with a four-fold higher affinity than thatobserved for ATP (Prodromou et al. (1997) Cell 90:65-75). It washypothesized that this difference was due to a lack of Mg²⁺ ions in theassay buffer, as Mg²⁺ has been demonstrated to be essential for ATP/ADPbinding to recombinant forms of the Hsp90 N-terminal nucleotide bindingdomain by Prodromou et al. (1997) Cell 90:65-75 and Obermann et al.(1998) J Cell Biol 143:901 -910.

[0369] This hypothesis was examined in experiments where the relativeaffinity of GRP94 for NECA, adenosine, ATP, ADP and AMP were compared inthe presence and absence of excess Mg²⁺ (FIG. 12). In these experiments,it was observed that although excess Mg²⁺ was without effect on thebinding of NECA or adenosine to GRP94, Mg²⁺ markedly stimulated thebinding of ATP, ADP and AMP. These data are consistent with recentcrystal structure data identifying Mg²⁺ interactions with the α and βphosphates as being requisite for ATP/ADP binding to the N-terminaldomain of Hsp90. See Prodromou et al. (1997) Cell 90:65-75. However,unlike the N-terminal domain of Hsp90, MgATP and MgADP bind to GRP94with nearly identical relative affinities. It should also be noted thatthe presence of excess Mg2+ was without effect on the relative bindingaffinities of cAMP and geldanamycin for GRP94.

Example 12 Nucleotide Requirement for Autophosphorylation and ATPHydrolysis

[0370] To test whether binding to the nucleotide binding pocket isdirectly responsible for the observed GRP94 autophosphorylationactivity, NECA and radicicol were utilized as inhibitors of ATP bindingto GRP94. Data regarding autophosphorylation activities are shown inFIG. 13A. In this experiment, the autophosphorylation activity of GRP94was assayed in the presence of NECA, radicicol, heparin and GTP. Heparinand GTP were included on the basis of previous studies indicating acasein kinase II-like contaminant in purified preparations of GRP94(Wearsch & Nicchitta (1997) J Biol Chem 272:5152-5156; Riera et al.(1999) Mol Cell Biochem 191:97-104; and Ramakrishnan et al. (1997) JCell Physiol 170:115-29). By similar logic, the relative effects ofthese compounds on GRP94 kinase activity were compared in parallel withpurified casein kinase II, with casein kinase II activity measured withpurified casein.

[0371] As is evident from the data presented in FIG. 13A, neither NECAnor radicicol, both of which bind to the N-terminal nucleotide bindingdomain of GRP94, significantly inhibit GRP94 derived or casein kinase IIactivity below the solvent background. Because of the relatively highhydrophobicity of NECA and radicicol, incubations containing thesecompounds contained significant concentrations of the ligand solvent,dimethylsulfoxide, which itself significantly reduced both theGRP94-derived and casein kinase II activities. Heparin and GTP markedlyattenuated GRP94-derived and casein kinase II activity. In summary,blocking nucleotide access to the N-terminal adenosine nucleotide GRP94binding pocket does not significantly inhibit GRP94 autophosphorylationactivity.

[0372] The findings that cycles of ATP binding and hydrolysis functionin the regulation of Hsp90 activity, and that GRP94 exhibits an ATPaseactivity suggest that GRP94 and Hsp90 are indeed regulated by a similarmechanism. To further evaluate this suggestion, the ATPase activity ofGRP94 was assayed as a function of ATP concentration (FIG. 13B). Twopoints are immediately evident from the observed data. First, the ATPaseactivity does not display saturation; no evidence for a Vmax could beobtained and so traditional criteria for enzymatic function (i.e.,Km/Kcat/Vmax) could not be applied. Secondly, the absolute magnitude ofthe ATPase activity exceeded the spontaneous rate of ATP hydrolysis byonly a small factor. The observed ATPase activity was sensitive toinhibition by NECA, and thus is likely generated upon binding of ATP tothe N-terminal nucleotide binding domain.

Example 13 Conformational Consequences of Adenosine Nucleotide Bindingto GRP94

[0373] Having been unable to identify a functional correlate of ATPbinding to GRP94, the effects of ATP, ADP, NECA and geldanamycin onGRP94 conformation were assessed. In these studies, the tryptophanemission spectra of GRP94, complexed with the indicated ligands, wasexamined as a measure of tertiary conformational state in accordancewith techniques described by Guilbault (1967) Fluoresence: Theory,Instrumentation, and Practice, Marcel Dekker, Inc., New York, N.Y. Asshown in FIG. 14, high concentrations of ATP or ADP elicited nearidentical changes in the GRP94 tryptophan fluorescence spectra.Significantly, in the presence of ATP or ADP, the tryptophanfluorescence was decreased, as was observed in the presence ofgeldanamycin. These data indicate that ATP and ADP elicit aconformational change similar to that occurring in the presence of theinhibitory ligand geldanamycin and that the conformation of GRP94 in theATP and ADP-bound state, as assessed by tryptophan fluorescence, areessentially identical. In contrast, the addition of NECA increased thetryptophan fluorescence, indicating that ligands can elicit differentconformational states in GRP94. As demonstrated in Examples 1-8 above,such changes in GRP94 conformation can have dramatic effects on GRP94chaperone function.

Summary of Examples 9-13

[0374] Examples 9-13 disclose that Hsp90 paralogs GRP94 and HSP90display distinct structural and functional interactions with adenosinenucleotides. Unlike HSP90, GRP94 displays specific, high affinitybinding interactions with substituted adenosine derivatives such asN-ethylcarboxamidoadenosine (NECA). In analyzing such interactions, theoccupancy states of the N-terminal ATP/ADP binding domains of GRP94 arecommunicated between the two identical subunits. This conclusion isdrawn from the observation that at saturation NECA is bound to GRP94 ata stoichiometry of 1 mol NECA:mol GRP94 dimer. In contrast to NECA, theGRP94 inhibitory ligand, radicicol, binds at a stoichiometry of 2mol:mol GRP94. Thus, although the relevant structural components of theadenosine nucleotide binding pocket are conserved between GRP94 andHsp90, the ligand specificities of the two binding sites differ. Thus,while it is not applicants' desire to be bound by a particularlymechanistic theory, it is envisioned that the specificity of ligandbinding to the N-terminal adenosine nucleotide binding pocket isinfluenced by the domains C and N-terminal to the binding pocket, wheresignificant sequence divergence between HSP90 and GRP94 can beidentified.

[0375] The data obtained from both traditional ligand binding studies(FIG. 9) and isothermal titration calorimetry demonstrate that GRP94binds NECA at a stoichiometry of 1 mol NECA: mol GRP94 dimer. Inaddition, competition studies indicate that NECA binding to GRP94 can bewholly competed by geldanamycin, radicicol, ATP, and ADP (FIGS.10A-10C), indicating that NECA is binding to the conserved, N-terminaladenosine nucleotide binding domain. Because GRP94 contains two suchsites per molecule (Wearsch & Nicchitta (1996b) Biochemistry35:16760-16769), it then follows that GRP94 subunits communicate withone another to confer single site occupancy.

[0376] The identification of ATP and ADP as the native ligands for theHsp90 proteins is based on crystallographic studies identifying anN-terminal, highly conserved nucleotide binding pocket (Prodromou et al.(1997) Cell 90:65-75), complementary in vivo studies demonstrating thatthe amino acids that participate in ATP/ADP binding are essential forHsp90 function in vivo and lastly (Obermann et al. (1998) J Cell Biol143:901-910; Panaretou et al. (1998) EMBO J 17:4829-4836), that theHsp90 proteins display ATPase activity (Grenert et al. (1999) J BiolChem 274:17525-17533; Nadeau et al. (1993) J Biol Chem 268:1479-1487;Obermann et al. (1998) J Cell Biol 143:901-910). That HSP90 and GRP94differ in NECA binding activity, despite the high homologies in theN-terminal nucleotide binding pockets of the two protein, suggests thatdifferences might also exist in the ability of the two proteins tocatalyze ATP hydrolysis. In fact, when the GRP94 ATPase activity wasinvestigated at ATP concentrations appropriate for such a low affinityinteraction it was observed that the GRP94 ATPase activity barelyexceeded the rate of spontaneous hydrolysis and, more importantly, didnot saturate at increasing ATP concentrations.

[0377] Further studies of the binding properties of the conserved domainindicated that it displays poor selectivity between adenosinenucleotides, and will bind ATP, dATP, ADP, AMP, cAMP and free adenosine.On the basis of these and other data, GRP94 conformation is regulated inan allosteric manner by an adenosine-bearing ligand other than ATP/ADP,based on ligand-mediated conformational regulation.

[0378] GRP94-dependent ATP hydrolysis, as displayed by the purifiedprotein in the absence of any, as yet unidentified co-factors, isnon-enzymatic, and therefore unlikely to contribute to the regulation ofGRP94 function. Further confounding the assignment of ATP and ADP as thephysiological ligands for GRP94 are the following observations. First,neither ATP nor ADP has been demonstrated to regulate GRP94 activity, asdescribed by Wearsch & Nicchitta (1997) J Biol Chem 272:5152-5156.Secondly, that by virtue of its insensitivity to NECA and radicicol, theGRP94 autophosphorylation activity does not reflect adenosine nucleotidebinding to the N-terminal nucleotide binding domain (FIG. 13). Thirdly,and perhaps most importantly, ATP, ADP, and the inhibitor geldanamycinelicit similar conformational changes in GRP94. Interestingly, in thepresence of NECA, a different conformational change from that occurringin the presence of ATP, ADP, or geldanamycin was observed (FIG. 14).These data are consistent with ATP and ADP binding to GRP94 andstabilizing the protein in an inactive conformation, as is observed inthe presence of geldanamycin.

[0379] In evaluating these data, the inability to identify an enzymaticbasis for the ATPase activity and the conformation data suggesting thatATP/ADP would serve as inhibitory agent, either unidentified accessoryproteins interact with GRP94 to substantively alter the kinetic andthermodynamic basis for its interaction with ATP/ADP or anadenosine-based ligand, other than ATP/ADP, serves as the physiologicalligand. The ligand is produced during times of cell stress, such asanoxia, nutrient deprivation or heat shock, to activate GRP94 function.The ligand elicits a conformational change in GRP94 that substantivelyalters its interaction with substrate (poly)peptides.

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What is claimed is:
 1. A method for purifying a complex comprising aGRP94 protein, the method comprising: (a) contacting a complexcomprising a GRP94 protein with a binding agent that preferentiallybinds GRP94, the binding agent immobilized to a solid phase support, toimmobilize the complex to the solid phase support; (b) collecting theremaining sample; and (c) eluting the complex from the solid phasesupport to give purified complex in the eluate.
 2. The method of claim1, wherein the binding agent is free of ATP or ADP.
 3. The method ofclaim 1, wherein the binding agent comprises a compound of formula (I):

or a compound of formula (II):

where: X and Y are the same or different and X and Y═C, N, O or S; and Xand Y can be substituted with hydrogen, hydroxyl, or oxygen, includingdouble-bonded oxygen; R¹=hydrogen, hydroxyl, C₁ to C₆ alkyl, C₁ to C₆branched alkyl, C₁ to C₆ hydroxyalkyl, branched C₁ to C₆ hydroxyalkyl,C₄ to C₈ cycloalkyl, C₁ to C₆ alkenyl, branched C₁ to C₆ alkenyl, C₄ toC₈ cycloalkenyl, C₄ to C₈ aryl, C₄ to C₈ aroyl, C₄ to C₈aryl-substituted C₁ to C₆ alkyl, C₁ to C₆ alkoxy, C₁ to C₆ branchedalkoxy, C₄ to C₈ aryloxy, primary, secondary or tertiary C₁ to C₆alkylamino, primary, secondary or tertiary branched C₁ to C₆ alkylamino,primary, secondary or tertiary cycloalkylamino, primary, secondary ortertiary C₄ to C₈ arylamino, C₁ to C₆ alkylcarboxylic acid, branched C₁to C₆ alkylcarboxylic acid, C₁ to C₆ alkylester, branched C₁ to C₆alkylester, C₄ to C₈ arylcarboxylic acid, C₄ to C₈ arlyester, C₄ to C₈aryl substituted C₁ to C₆ alkyl, C₄ to C₁₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring,alkyl-substituted or aryl-substituted C₄ to C₁₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring; or hydroxyl-,amino-, or halo-substituted versions thereof; or R¹ is halo where halois chloro, fluoro, bromo, or iodo; R²=hydrogen, hydroxyl, C₁ to C₆alkyl, C₁ to C₆ branched alkyl, C₁ to C₆ hydroxyalkyl, branched C₁ to C₆hydroxyalkyl, C₄ to C₈ cycloalkyl, C₁ to C₆ alkenyl, branched C₁ to C₆alkenyl, C₄ to C₈ cycloalkenyl, C₄ to C₈ aryl, C₄ to C₈ aroyl, C₄ to C₈aryl-substituted C₁ to C₆ alkyl, C₁ to C₆ alkoxy, C₁ to C₆ branchedalkoxy, C₄ to C₈ aryloxy, primary, secondary or tertiary C₁ to C₆alkylamino, primary, secondary or tertiary branched C₁ to C₆ alkylamino,primary, secondary or tertiary cycloalkylamino, primary, secondary ortertiary C₄ to C₈ arylamino, C₁ to C₆ alkylcarboxylic acid, branched C₁to C₆ alkylcarboxylic acid, C₁ to C₆ alkylester, branched C₁ to C₆alkylester, C₄ to C₈ arylcarboxylic acid, C₄ to C₈ arlyester, C₄ to C₈aryl substituted C₁ to C₆ alkyl, C₄ to C₁₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring,alkyl-substituted or aryl-substituted C₄ to C₁₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring; or hydroxyl-,amino-, or halo-substituted versions thereof; or R² is halo where halois chloro, fluoro, bromo, or iodo; R³=hydrogen, hydroxyl, C₁ to C₆alkyl, C₁ to C₆ branched alkyl, C₁ to C₆ hydroxyalkyl, branched C₁ to C₆hydroxyalkyl, C₄ to C₈ cycloalkyl, C₁ to C₆ alkenyl, branched C₁ to C₆alkenyl, C₄ to C₈ cycloalkenyl, C₄ to C₈ aryl, C₄ to C₈ aroyl, C₄ to C₈aryl-substituted C₁ to C₆ alkyl, C₁ to C₆ alkoxy, C₁ to C₆ branchedalkoxy, C₄ to C₈ aryloxy, primary, secondary or tertiary C₁ to C₆alkylamino, primary, secondary or tertiary branched C₁ to C₆ alkylamino,primary, secondary or tertiary cycloalkylamino, primary, secondary ortertiary C₄ to C₈ arylamino, C₁ to C₆ alkylcarboxylic acid, branched C₁to C₆ alkylcarboxylic acid, C₁ to C₆ alkylester, branched C₁ to C₆alkylester, C₄ to C₈ arylcarboxylic acid, C₄ to C₈ arlyester, C₄ to C₈aryl substituted C₁ to C₆ alkyl, C₄ to C₁₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring,alkyl-substituted or aryl-substituted C₄ to C₁₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring; or hydroxyl-,amino-, or halo-substituted versions thereof; or R³ is halo where halois chloro, fluoro, bromo, or iodo; and R⁴=C, to C₆ alkyl, C₁ to C₆branched alkyl, C₄ to C₈ cycloalkyl with or without O, N or S in thering, C₁ to C₆ alkenyl, branched C₁ to C₆ alkenyl, C₄ to C₈ cycloalkenylwith or without O, N or S in the ring, C₄ to C₈ aroyl, C₄ to C₈ aryl, C₄to C₁₂ heterocyclic or heteropolycyclic alkyl or aryl with O, N or S inthe ring, C₄ to C₈ aryl-substituted C₁ to C₆ alkyl, alkyl-substituted oraryl-substituted C₄ to C₁₂ heterocyclic or heteropolycyclic alkyl oraryl with O, N or S in the ring, alkyl-substituted C₄ to C₈ aroyl, oralkyl-substituted C₄ to C₈ aryl; or hydroxyl-, amino-, orhalo-substituted versions thereof where halo is chloro, bromo, fluoro oriodo.
 4. The method of claim 1, wherein the complex further comprises anantigenic molecule.
 5. The method of claim 1, wherein the complex isfrom a warm-blooded vertebrate.
 6. The method of claim 5, wherein thecomplex is from a mammal.
 7. The method of claim 6, wherein the mammalis selected from the group consisting of human, mouse, pig, rat, ape,monkey, cat, guinea pig, cow, goat, and horse.
 8. The method of claim 1,wherein the complex is produced in vitro.
 9. The method of claim 1,wherein the complex is purified from cancerous tissue.
 10. The method ofclaim 9, wherein the complex is purified from cancerous tissueautologous to a vertebrate subject to be treated with the complex. 11.The method of claim 9, wherein the complex is purified from canceroustissue from a second vertebrate subject that is the same type as acancerous tissue present in a first vertebrate subject to be treatedwith the composition.
 12. The method of claim 9, wherein the complex isobtained from a tumor cell line of a type of cancer.
 13. The method ofclaim 9, wherein the cancerous tissue comprises comprises a sarcoma orcarcinoma, selected from the group consisting of fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma, leukemia, lymphoma, multiple myeloma, Waldenströom'smacroglobulinemia, and heavy chain disease.
 14. The method of claim 1,wherein the complex is purified from a eukaryotic cell infected with apathogen that causes an infectious disease.
 15. The method of claim 14,wherein the infectious disease is caused by a pathogen selected from thegroup consisting of viruses, bacteria, fungi, protozoa, and parasites.16. The method of claim 15, wherein the viral pathogen is selected fromthe group consisting of hepatitis type A, hepatitis type B, hepatitistype C, influenza, varicella, adenovirus, herpes simplex type I (HSV-I),herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus,rotavirus, respiratory syncytial virus (RSV), papilloma virus, papovavirus, cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackievirus, mumps virus, measles virus, rubella virus, polio virus, humanimmunodeficiency virus type I (HIV-I), and human immunodeficiency virustype II (HIV-II).
 17. The method of claim 15, wherein the bacterialpathogen is selected from the group consisting of Mycobacteria,Rickettsia, Mycoplasma, Neisseria, and Legionella.
 18. The method ofclaim 15, wherein the protozoal pathogen is selected from the groupconsisting of Leishmania, Kokzidioa, Trypanosoma, Chlamydia, andRickettsia.
 19. The method of claim 1, wherein complex bound to theimmobilized binding agent is eluted by washing the solid phase supportwith a buffer comprising the binding agent to give complex in theeluate.
 20. A product produced by the method of claim
 1. 21. A methodfor isolating an antigenic molecule associated with a GRP94 complex, themethod comprising: (A) contacting a complex comprising GRP94 and anantigenic molecule with a binding agent that preferentially binds GRP94,the binding agent immobilized to a solid phase support, to immobilizethe complex to the solid phase support; (b) collecting the remainingsample; (c) eluting the complex from the solid phase support to givepurified complex in the eluate; and (d) isolating the antigenic moleculefrom the eluate.
 22. The method of claim 21, wherein the binding agentis free of ATP or ADP.
 23. The method of claim 21, wherein the complexcomprising GRP94 and the antigenic molecule is from a warm-bloodedvertebrate.
 24. The method of claim 23, wherein the complex comprisingGRP94 and the antigenic molecule is from a mammal.
 25. The method ofclaim 24, wherein the mammal is selected from the group consisting ofhuman, mouse, pig, rat, ape, monkey, cat, guinea pig, cow, goat, andhorse.
 26. The method of claim 21, wherein the complex comprising GRP94and the antigenic molecule is produced in vitro.
 27. The method of claim21, wherein the antigenic molecule is an exogenous antigenic peptide.28. The method of claim 21, wherein the antigenic molecule is a peptidewith which the GRP94 complex is endogenously associated in vivo.
 29. Themethod of claim 21, wherein the complex comprising GRP94 and theantigenic molecule is isolated from cancerous tissue.
 30. The method ofclaim 29, wherein the complex comprising GRP94 and the antigenicmolecule is isolated from cancerous tissue autologous to a vertebratesubject to be treated with the complex comprising GRP94 and theantigenic molecule.
 31. The method of claim 29, wherein the complexcomprising GRP94 and the antigenic molecule is isolated from canceroustissue from a second vertebrate subject that is the same type as acancerous tissue present in a first vertebrate subject to be treatedwith the complex comprising GRP94 and the antigenic molecule.
 32. Themethod of claim 29, wherein the complex comprising GRP94 and theantigenic molecule is obtained from a tumor cell line of a type ofcancer.
 33. The method of claim 29, wherein the cancerous tissuecomprises a sarcoma or carcinoma, selected from the group consisting offibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma, leukemia, lymphoma, multiple myeloma, Waldenströom'smacroglobulinemia, and heavy chain disease.
 34. The method of claim 21,wherein the antigenic molecule is an antigenic peptide that is presentin a eukaryotic cell infected with a pathogen which causes saidinfectious disease but not present in said eukaryotic cell when saideukaryotic cell is not infected with said pathogen.
 35. The method ofclaim 34, wherein said infectious disease is caused by a pathogenselected from the group consisting of viruses, bacteria, fungi,protozoa, and parasites.
 36. The method of claim 35, wherein said viralpathogen is selected from the group consisting of hepatitis type A,hepatitis type B, hepatitis type C, influenza, varicella, adenovirus,herpes simplex type I (HSV-I), herpes simplex type II (HSV-II),rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytialvirus (RSV), papilloma virus, papova virus, cytomegalovirus,echinovirus, arbovirus, huntavirus, coxsackie virus, mumps virus,measles virus, rubella virus, polio virus, human immunodeficiency virustype I (HIV-I), and human immunodeficiency virus type II (HIV-II). 37.The method of claim 35, wherein said bacterial pathogen is selected fromthe group consisting of Mycobacteria, Mycoplasma, Neisseria, andLegionella.
 38. The method of claim 35, wherein said protozoal pathogenis selected from the group consisting of Leishmania, Kokzidioa,Trypanosoma, Chlamydia, and Rickettsia.
 39. The method of claim 21,wherein the binding agent comprises a compound of formula (I):

or a compound of formula (Il):

where: X and Y are the same or different and X and Y═C, N, O or S; and Xand Y can be substituted with hydrogen, hydroxyl, or oxygen, includingdouble-bonded oxygen; R¹=hydrogen, hydroxyl, C₁ to C₆ alkyl, C₁ to C₆branched alkyl, C₁ to C₆ hydroxyalkyl, branched C₁ to C₆ hydroxyalkyl,C₄ to C₈ cycloalkyl, C₁ to C₆ alkenyl, branched C₁ to C₆ alkenyl, C₄ toC₈ cycloalkenyl, C₄ to C₈ aryl, C₄ to C₈ aroyl, C₄ to C₈aryl-substituted C₁ to C₆ alkyl, C₁ to C₆ alkoxy, C₁ to C₆ branchedalkoxy, C₄ to C₈ aryloxy, primary, secondary or tertiary C₁ to C₆alkylamino, primary, secondary or tertiary branched C₁ to C₆ alkylamino,primary, secondary or tertiary cycloalkylamino, primary, secondary ortertiary C₄ to C₈ arylamino, C₁ to C₆ alkylcarboxylic acid, branched C₁to C₆ alkylcarboxylic acid, C₁ to C₆ alkylester, branched C₁ to C₆alkylester, C₄ to C₈ arylcarboxylic acid, C₄ to C₈ arlyester, C₄ to C₈aryl substituted C₁ to C₆ alkyl, C₄ to C₁₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring,alkyl-substituted or aryl-substituted C₄ to C₁₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring; or hydroxyl-,amino-, or halo-substituted versions thereof; or R¹ is halo where halois chloro, fluoro, bromo, or iodo; R²=hydrogen, hydroxyl, C₁ to C₆alkyl, C₁ to C₆ branched alkyl, C₁ to C₆ hydroxyalkyl, branched C₁ to C₆hydroxyalkyl, C₄ to C₈ cycloalkyl, C₁ to C₆ alkenyl, branched C₁ to C₆alkenyl, C₄ to C₈ cycloalkenyl, C₄ to C₈ aryl, C₄ to C₈ aroyl, C₄ to C₈aryl-substituted C₁ to C₆ alkyl, C₁ to C₆ alkoxy, C₁ to C₆ branchedalkoxy, C₄ to C₈ aryloxy, primary, secondary or tertiary C₁ to C₆alkylamino, primary, secondary or tertiary branched C₁ to C₆ alkylamino,primary, secondary or tertiary cycloalkylamino, primary, secondary ortertiary C₄ to C₈ arylamino, C₁ to C₆ alkylcarboxylic acid, branched C₁to C₆ alkylcarboxylic acid, C₁ to C₆ alkylester, branched C₁ to C₆alkylester, C₄ to C₈ arylcarboxylic acid, C₄ to C₈ arlyester, C₄ to C₈aryl substituted C₁ to C₆ alkyl, C₄ to C₁₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring,alkyl-substituted or aryl-substituted C₄ to C₁₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring; or hydroxyl-,amino-, or halo-substituted versions thereof; or R² is halo where halois chloro, fluoro, bromo, or iodo; R³=hydrogen, hydroxyl, C₁ to C₆alkyl, C₁ to C₆ branched alkyl, C₁ to C₆ hydroxyalkyl, branched C₁ to C₆hydroxyalkyl, C₄ to C₈ cycloalkyl, C₁ to C₆ alkenyl, branched C₁ to C₆alkenyl, C₄ to C₈ cycloalkenyl, C₄ to C₈ aryl, C₄ to C₈ aroyl, C₄ to C₈aryl-substituted C₁ to C₆ alkyl, C₁ to C₆ alkoxy, C₁ to C₆ branchedalkoxy, C₄ to C₈ aryloxy, primary, secondary or tertiary C₁ to C₆alkylamino, primary, secondary or tertiary branched C₁ to C₆ alkylamino,primary, secondary or tertiary cycloalkylamino, primary, secondary ortertiary C₄ to C₈ arylamino, C₁ to C₆ alkylcarboxylic acid, branched C₁to C₆ alkylcarboxylic acid, C₁ to C₆ alkylester, branched C₁ to C₆alkylester, C₄ to C₈ arylcarboxylic acid, C₄ to C₈ arlyester, C₄ to C₈aryl substituted C₁ to C₆ alkyl, C₄ to C₁₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring,alkyl-substituted or aryl-substituted C₄ to C₁₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring; or hydroxyl-,amino-, or halo-substituted versions thereof; or R³ is halo where halois chloro, fluoro, bromo, or iodo; and R⁴=C₁ to C₆ alkyl, C₁ to C₆branched alkyl, C₄ to C₈ cycloalkyl with or without O, N or S in thering, C₁ to C₆ alkenyl, branched C₁ to C₆ alkenyl, C₄ to C₈ cycloalkenylwith or without O, N or S in the ring, C₄ to C₈ aroyl, C₄ to C₈ aryl, C₄to C₁₂ heterocyclic or heteropolycyclic alkyl or aryl with O, N or S inthe ring, C₄ to C₈ aryl-substituted C₁ to C₆ alkyl, alkyl-substituted oraryl-substituted C₄ to C₁₂ heterocyclic or heteropolycyclic alkyl oraryl with O, N or S in the ring, alkyl-substituted C₄ to C₈ aroyl, oralkyl-substituted C₄ to C₈ aryl; or hydroxyl-, amino-, orhalo-substituted versions thereof where halo is chloro, bromo, fluoro oriodo.
 40. The method of claim 21, wherein complex bound to theimmobilized binding agent is eluted by washing the solid phase supportwith a buffer comprising the binding agent to give complex in theeluate.
 41. A product produced by the method of claim
 21. 42. A methodfor detecting a complex comprising GRP94 in a sample suspected ofcontaining a complex comprising GRP94, the method comprising: (A)contacting the sample with a binding agent that preferentially bindsGRP94 under conditions favorable to binding a complex comprising GRP94to the binding substance to form a second complex there between; and (b)detecting the second complex via a label conjugated to the bindingsubstance or via a labeled reagent that specifically binds to the secondcomplex subsequent to its formation.
 43. The method of claim 42, whereinthe binding substance is conjugated with a detectable label and whereindetecting step (b) comprises: (i) separating the second complex fromunbound labeled binding substance; and (ii) detecting the detectablelabel which is present in the second complex or which is unbound. 44.The method of claim 42, wherein the binding substance is free of ATP orADP.
 45. The method of claim 42, wherein the binding agent comprises acompound of formula (I):

or a compound of formula (II):

where: X and Y are the same or different and X and Y═C, N, O or S; and Xand Y can be substituted with hydrogen, hydroxyl, or oxygen, includingdouble-bonded oxygen; R¹=hydrogen, hydroxyl, C₁ to C₆ alkyl, C₁ to C₆branched alkyl, C₁ to C₆ hydroxyalkyl, branched C₁ to C₆ hydroxyalkyl,C₄ to C₈ cycloalkyl, C₁ to C₆ alkenyl, branched C₁ to C₆ alkenyl, C₄ toC₈ cycloalkenyl, C₄ to C₈ aryl, C₄ to C₈ aroyl, C₄ to C₈aryl-substituted C₁ to C₆ alkyl, C₁ to C₆ alkoxy, C₁ to C₆ branchedalkoxy, C₄ to C₈ aryloxy, primary, secondary or tertiary C₁ to C₆alkylamino, primary, secondary or tertiary branched C₁ to C₆ alkylamino,primary, secondary or tertiary cycloalkylamino, primary, secondary ortertiary C₄ to C₈ arylamino, C₁ to C₆ alkylcarboxylic acid, branched C₁to C₆ alkylcarboxylic acid, C₁ to C₆ alkylester, branched C₁ to C₆alkylester, C₄ to C₈ arylcarboxylic acid, C₄ to C₈ arlyester, C₄ to C₈aryl substituted C₁ to C₆ alkyl, C₄ to C₁₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring,alkyl-substituted or aryl-substituted C₄ to C₁₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring; or hydroxyl-,amino-, or halo-substituted versions thereof; or R¹ is halo where halois chloro, fluoro, bromo, or iodo; R²=hydrogen, hydroxyl, C₁ to C₆alkyl, C₁ to C₆ branched alkyl, C₁ to C₆ hydroxyalkyl, branched C₁ to C₆hydroxyalkyl, C₄ to C₈ cycloalkyl, C₁ to C₆ alkenyl, branched C₁ to C₆alkenyl, C₄ to C₈ cycloalkenyl, C₄ to C₈ aryl, C₄ to C₈ aroyl, C₄ to C₈aryl-substituted C₁ to C₆ alkyl, C₁ to C₆ alkoxy, C₁ to C₆ branchedalkoxy, C₄ to C₈ aryloxy, primary, secondary or tertiary C₁ to C₆alkylamino, primary, secondary or tertiary branched C₁ to C₆ alkylamino,primary, secondary or tertiary cycloalkylamino, primary, secondary ortertiary C₄ to C₈ arylamino, C₁ to C₆ alkylcarboxylic acid, branched C₁to C₆ alkylcarboxylic acid, C₁ to C₆ alkylester, branched C₁ to C₆alkylester, C₄ to C₈ arylcarboxylic acid, C₄ to C₈ arlyester, C₄ to C₈aryl substituted C₁ to C₆ alkyl, C₄ to C₁₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring,alkyl-substituted or aryl-substituted C₄ to C₁₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring; or hydroxyl-,amino-, or halo-substituted versions thereof; or R² is halo where halois chloro, fluoro, bromo, or iodo; R³=hydrogen, hydroxyl, C₁ to C₆alkyl, C₁ to C₆ branched alkyl, C₁ to C₆ hydroxyalkyl, branched C₁ to C₆hydroxyalkyl, C₄ to C₈ cycloalkyl, C₁ to C₆ alkenyl, branched C₁ to C₆alkenyl, C₄ to C₈ cycloalkenyl, C₄ to C₈ aryl, C₄ to C₈ aroyl, C₄ to C₈aryl-substituted C₁ to C₆ alkyl, C₁ to C₆ alkoxy, C₁ to C₆ branchedalkoxy, C₄ to C₈ aryloxy, primary, secondary or tertiary C₁ to C₆alkylamino, primary, secondary or tertiary branched C₁ to C₆ alkylamino,primary, secondary or tertiary cycloalkylamino, primary, secondary ortertiary C₄ to C₈ arylamino, C₁ to C₆ alkylcarboxylic acid, branched C₁to C₆ alkylcarboxylic acid, C₁ to C₆ alkylester, branched C₁ to C₆alkylester, C₄ to C₈ arylcarboxylic acid, C₄ to C₈ arlyester, C₄ to C₈aryl substituted C₁ to C₆ alkyl, C₄ to C₁₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring,alkyl-substituted or aryl-substituted C₄ to C₁₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring; or hydroxyl-,amino-, or halo-substituted versions thereof; or R³ is halo where halois chloro, fluoro, bromo, or iodo; and R⁴=C₁ to C₆ alkyl, C₁ to C₆branched alkyl, C₄ to C₈ cycloalkyl with or without O, N or S in thering, C₁ to C₆ alkenyl, branched C₁ to C₆ alkenyl, C₄ to C₈ cycloalkenylwith or without O, N or S in the ring, C₄ to C₈ aroyl, C₄ to C₈ aryl, C₄to C₁₂ heterocyclic or heteropolycyclic alkyl or aryl with O, N or S inthe ring, C₄ to C₈ aryl-substituted C₁ to C₆ alkyl, alkyl-substituted oraryl-substituted C₄ to C₁₂ heterocyclic or heteropolycyclic alkyl oraryl with O, N or S in the ring, alkyl-substituted C₄ to C₈ aroyl, oralkyl-substituted C₄ to C₈ aryl; or hydroxyl-, amino-, orhalo-substituted versions thereof where halo is chloro, bromo, fluoro oriodo.
 46. The method of claim 42, wherein the complex comprising GRP94further comprises an antigenic molecule.
 47. The method of claim 42,wherein the complex comprising GRP94 is from a warm-blooded vertebrate.48. The method of claim 47, wherein the complex comprising GRP94 is froma mammal.
 49. The method of claim 48, wherein the mammal is selectedfrom the group consisting of human, mouse, pig, rat, ape, monkey, cat,guinea pig, cow, goat, and horse.
 50. A kit for detecting, isolating orpurifying a complex comprising GRP94 or an antigenic molecule associatedwith a complex comprising GRP94, the kit comprising: (a) a binding agentthat preferentially binds GRP94 contained in a first container; and (b)an elution buffer for use in eluting a complex comprising GRP94 from thebinding agent, the elution buffer contained in a second container. 51.The kit of claim 50, further comprising a solid phase support containedin a second container.
 52. The kit of claim 50, wherein the bindingagent is immobilized to a solid phase support.
 53. The kit of claim 50,wherein the binding agent is free of ATP or ADP.
 54. The kit of claim50, further comprising an elution buffer for use in eluting an antigenicmolecule from a complex comprising GRP94, the elution buffer containedin a fourth container.
 55. The kit of claim 50, wherein the bindingagent comprises a detectable label or indicator.
 56. The kit of claim50, further comprising a reagent or indicator that comprises adetectable label, the indicator contained in a fifth container.
 57. Thekit of claim 56, wherein the indicator is a radioactive label or anenzyme.
 58. A method of screening a candidate substance for an abilityto modulate GRP94 biological activity, the method comprising: (a)establishing a test sample comprising a GRP94 protein and a ligand for aGRP94 protein; (b) administering a candidate substance to the testsample; and (c) measuring the effect of the candidate substance onbinding of the GRP94 protein and the ligand in the test sample tothereby determine the ability of the candidate substance to modulate thebiological activity of the GRP94 protein.
 59. The method of claim 58,wherein the test sample further comprises an indicator, and the abilityof the candidate substance to modulate biological activity of the GRP94protein is determined by: (i) detecting a signal produced by theindicator upon an effect of the candidate substance on binding of theGRP94 protein and the ligand for the GRP94 protein; and (ii) identifyingthe candidate substance as a modulator of the biological activity of theGRP94 protein based upon an amount of signal produced as compared to acontrol sample.
 60. The method of claim 58, wherein the ligand is alsoan indicator.
 61. The method of claim 60, wherein the ligand is 8-ANS.62. The method of claim 58, wherein the candidate substance is furthercharacterized as a candidate polypeptide, and further comprising thestep of purifying and isolating a nucleic acid molecule encoding thecandidate polypeptide.
 63. A method for a identifying a candidatesubstance as an activator of the biological activity of a Hsp90 protein,the method comprising: (a) establishing a test sample comprising a Hsp90protein and a candidate substance; (b) administering 8-ANS to the testsample; and (c) detecting a fluorescence signal produced by the 8-ANS;and (d) identifying the candidate substance as an activator of thebiological activity of a Hsp90 protein based upon an amount offluorescence signal produced by the 8-ANS as compared to a controlsample.
 64. The method of claim 65, wherein the Hsp90 protein is GRP94.65. The method of claim 63, further comprising incubating the Hsp90protein with the candidate substance at 37° C. for about one hour priorto adding the 8-ANS.
 66. The method of claim 63, wherein the 8-ANS isadded in an approximately equimolar amount to the Hsp90 protein.
 67. Themethod of claim 63, wherein the candidate substance is identified as anactivator of the biological activity of a Hsp90 protein by detection ofan increased 8-ANS fluorescence signal as compared to a control sample68. A method for a identifying a candidate substance as an inhibitor ofthe biological activity of a Hsp90 protein, the method comprising: (a)establishing a test sample comprising a Hsp90 protein and a candidatesubstance; (b) heat-shocking the test sample to induce a conformationalchange to the Hsp90 protein; (c) administering 8-ANS to the test sample;and (d) detecting a fluorescence signal produced by binding of 8-ANS tothe Hsp90 protein; and (e) identifying the candidate substance as aninhibitor of the biological activity of a Hsp90 protein based upon anamount of fluorescence signal produced by the 8-ANS as compared to acontrol sample.
 69. The method of claim 68, wherein the Hsp90 protein isGRP94.
 70. The method of claim 68, further comprising incubating thetest sample at 37° C. for about one hour prior to heat-shocking the testsample.
 71. The method of claim 69, where the heat-shocking is carriedout at 50° C. for about 15 minutes.
 72. The method of claim 68, whereinthe 8-ANS is added in an approximately equimolar amount to the Hsp90protein.
 73. The method of claim 68, wherein the candidate substance isidentified as an inhibitor of the biological activity of a Hsp90 proteinby detection of a decreased 8-ANS fluorescence signal as compared to acontrol sample.
 74. A method of modulating the biological activity of aHsp90 protein, the method comprising contacting a Hsp90 protein with aneffective amount of a Hsp90 protein activity-modulating substance tothereby modulate the biological activity.
 75. The method of claim 74,wherein the Hsp90 protein is GRP94.
 76. The method of claim 75, whereinthe GRP94 protein is in an in vitro sample.
 77. The method of claim 75,wherein the GRP94 protein is in a cell.
 78. The method of claim 77,wherein the cell is a tumor cell or is a cell infected with a pathogen.79. The method of claim 77, wherein the cell is in a subject.
 80. Amethod of treating a subject suffering from a disorder whereinmodulation of the biological activity of a GRP94 protein is desirable,the method comprising administering to the subject an effective amountof a GRP94 protein modulator, whereby modulation of the biologicalactivity of a GRP94 protein in the subject is accomplished.
 81. Themethod of claim 80, wherein GRP94 biological activity that is modulatedis eliciting an immune response in a subject, protein transport from theendoplasmic reticulum, recovery from hypoxic/anoxic stress, recoveryfrom nutrient deprivation, recovery from heat stress, or combinationsthereof.
 82. The method of claim 80, wherein the disorder is a type ofcancer, an infectious disease, a disorder associated with impairedprotein transport from the endoplasmic reticulum, a disorder associatedwith ischemia/reperfusion, or combinations thereof.
 83. The method ofclaim 82, further comprising administering to the subject a compositioncomprising a therapeutically or prophylactically effective amount of apurified complex, said complex comprising a GRP94 protein bound to anantigenic molecule specific to said disorder.
 84. The method of claim 82wherein the disorder associated with ischemia/reperfusion is a result ofcardiac arrest, asystole and sustained ventricular arrythmias, cardiacsurgery, cardiopulmonary bypass surgery, organ transplantation, spinalcord injury, head trauma, stroke, thromboembolic stroke, hemorrhagicstroke, cerebral vasospasm, hypotension, hypoglycemia, statusepilepticus, an epileptic seizure, anxiety, schizophrenia, aneurodegenerative disorder, Alzheimer's disease, Huntington's disease,amyotrophic lateral sclerosis (ALS), or neonatal stress.
 85. A methodfor altering a subsequent cellular response to an ischemic condition ata tissue location in a subject, the method comprising treating the cellsat the tissue location with a GRP94 protein modulator whereby GRP94activity in cells is enhanced to a degree effective to alter asubsequent cellular response to an ischemic condition.
 86. The method ofclaim 85 wherein the subsequent ischemic condition is the result ofcardiac arrest, asystole and sustained ventricular arrythmias, cardiacsurgery, cardiopulmonary bypass surgery, organ transplantation, spinalcord injury, head trauma, stroke, thromboembolic stroke, hemorrhagicstroke, cerebral vasospasm, hypotension, hypoglycemia, statusepilepticus, an epileptic seizure, anxiety, schizophrenia, aneurodegenerative disorder, Alzheimer's disease, Huntington's disease,amyotrophic lateral sclerosis (ALS), or neonatal stress.
 87. The methodof claim 85, wherein the tissue locus comprises donor tissue fortransplantation.
 88. A pharmaceutical composition comprising atherapeutically effective amount of a modulator of a biological activityof a GRP94 protein, and a pharmaceutically acceptable diluent orvehicle.
 89. The composition of claim 88, further comprising a compoundof formula (I):

where: X and Y are the same or different and X and Y═C₁ N, O or S; and Xand Y can be substituted with hydrogen, hydroxyl, or oxygen, includingdouble-bonded oxygen; R¹=hydrogen, hydroxyl, C₁ to C₆ alkyl, C₁ to C₆branched alkyl, C₁ to C₆ hydroxyalkyl, branched C₁ to C₆ hydroxyalkyl,C₄ to C₈ cycloalkyl, C₁ to C₆ alkenyl, branched C₁ to C₆ alkenyl, C₄ toC₈ cycloalkenyl, C₄ to C₈ aryl, C₄ to C₈ aroyl, C₄ to C₈aryl-substituted C₁ to C₆ alkyl, C₁ to C₆ alkoxy, C₁ to C₆ branchedalkoxy, C₄ to C₈ aryloxy, primary, secondary or tertiary C₁ to C₆alkylamino, primary, secondary or tertiary branched C₁ to C₆ alkylamino,primary, secondary or tertiary cycloalkylamino, primary, secondary ortertiary C₄ to C₈ arylamino, C₁ to C₆ alkylcarboxylic acid, branched C₁to C₆ alkylcarboxylic acid, C₁ to C₆ alkylester, branched C₁ to C₆alkylester, C₄ to C₈ arylcarboxylic acid, C₄ to C₈ arlyester, C₄ to C₈aryl substituted C₁ to C₆ alkyl, C₄ to C₁₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring,alkyl-substituted or aryl-substituted C₄ to C₁₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring; or hydroxyl-,amino-, or halo-substituted versions thereof; or R¹ is halo where halois chloro, fluoro, bromo, or iodo; R²=hydrogen, hydroxyl, C₁ to C₆alkyl, C₁ to C₆ branched alkyl, C₁ to C₆ hydroxyalkyl, branched C₁ to C₆hydroxyalkyl, C₄ to C₈ cycloalkyl, C₁ to C₆ alkenyl, branched C₁ to C₆alkenyl, C₄ to C₈ cycloalkenyl, C₄ to C₈ aryl, C₄ to C₈ aroyl, C₄ to Caaryl-substituted C₁ to C₆ alkyl, C₁ to C₆ alkoxy, C₁ to C₆ branchedalkoxy, C₄ to C₈ aryloxy, primary, secondary or tertiary C₁ to C₆alkylamino, primary, secondary or tertiary branched C₁ to C₆ alkylamino,primary, secondary or tertiary cycloalkylamino, primary, secondary ortertiary C₄ to C₈ arylamino, C₁ to C₆ alkylcarboxylic acid, branched C₁to C₆ alkylcarboxylic acid, C₁ to C₆ alkylester, branched C₁ to C₆alkylester, C₄ to C₈ arylcarboxylic acid, C₄ to C₈ arlyester, C₄ to C₈aryl substituted C₁ to C₆ alkyl, C₄ to C₁₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring,alkyl-substituted or aryl-substituted C₄ to C₁₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring; or hydroxyl-,amino-, or halo-substituted versions thereof; or R² is halo where halois chloro, fluoro, bromo, or iodo; and R³=hydrogen, hydroxyl, C₁ to C₆alkyl, C₁ to C₆ branched alkyl, C₁ to C₆ hydroxyalkyl, branched C₁ to C₆hydroxyalkyl, C₄ to C₈ cycloalkyl, C₁ to C₆ alkenyl, branched C₁ to C₆alkenyl, C₄ to C₈ cycloalkenyl, C₄ to C₈ aryl, C₄ to C₈ aroyl, C₄ to C₈aryl-substituted C₁ to C₆ alkyl, C₁ to C₆ alkoxy, C₁ to C₆ branchedalkoxy, C₄ to C₈ aryloxy, primary, secondary or tertiary C₁ to C₆alkylamino, primary, secondary or tertiary branched C₁ to C₆ alkylamino,primary, secondary or tertiary cycloalkylamino, primary, secondary ortertiary C₄ to C₈ arylamino, C₁ to C₆ alkylcarboxylic acid, branched, C₁to C₆ alkylcarboxylic acid, C₁ to C₆ alkylester, branched C₁ to C₆alkylester, C₄ to C₈ arylcarboxylic acid, C₄ to C₈ arlyester, C₄ to C₈aryl substituted C₁ to C₆ alkyl, C₄ to C₁₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring,alkyl-substituted or aryl-substituted C₄ to C₁₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring; or hydroxyl-,amino-, or halo-substituted versions thereof; or R³ is halo where halois chloro, fluoro, bromo, or iodo.
 90. The composition of claim 88,wherein the therapeutically effective amount of the modulator rangesfrom about 0.01 mg to about 10,000 mg.
 91. The composition of claim 90,wherein the therapeutically effective amount of the modulator rangesfrom about 0.1 mg to about 1,000 mg.
 92. The composition of claim 91,wherein the therapeutically effective amount of the modulator rangesfrom about 1 mg to about 300 mg.
 93. A method for preparing animmunogenic composition for inducing an immune response in a vertebratesubject, the method comprising: (a) harvesting from a eukaryotic cell animmunogenic complex comprising a Hsp90 protein non-covalently bound toan antigenic molecule, said complex, when administered to saidvertebrate subject being operative at initiating an immune response insaid vertebrate subject, wherein said eukaryotic cell has been treatedwith an activating ligand of a Hsp90 protein; and (b) combining saidcomplex with a pharmaceutically acceptable carrier.
 94. The method ofclaim 93, wherein the Hsp90 protein is GRP94 or HSP90.
 95. The method ofclaim 93, wherein the antigenic molecule is a peptide with which theHsp90 protein is endogenously associated in vivo.
 96. The method ofclaim 93, wherein the complex is harvested from a cell of a type ofcancer.
 97. The method of claim 96, wherein the cell from the type ofcancer is isolated from a vertebrate subject.
 98. The method of claim97, wherein the cell from the type of cancer is isolated from canceroustissue autologous to a vertebrate subject to be treated with theimmunogenic composition.
 99. The method of claim 97, wherein the cellfrom the type of cancer is isolated from cancerous tissue from a secondvertebrate subject that is the same type as a cancerous tissue presentin a first vertebrate subject to be treated with the immunogeniccomposition.
 100. The method of claim 96, wherein the cell is obtainedfrom a tumor cell line of said type of cancer.
 101. The method of claim96, wherein the cancer comprises a sarcoma or carcinoma, selected fromthe group consisting of fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma, leukemia, lymphoma, multiple myeloma, Waldenströom'smacroglobulinemia, and heavy chain disease.
 102. The method of claim 93,wherein the eukaryotic cell has been transfected with a nucleic acidconstruct encoding the antigenic molecule, whereby the antigenicmolecule is expressed in the eukaryotic cell.
 103. The method of claim93, wherein the eukaryotic cell comprises a cell infected with apathogen.
 104. The method of claim 103, wherein the antigenic moleculeis an antigenic peptide that is present in said eukaryotic cell infectedwith said pathogen but not present in said eukaryotic cell when saideukaryotic cell is not infected with said pathogen.
 105. The method ofclaim 103, wherein said pathogen is selected from the group consistingof viruses, bacteria, fungi, protozoa, and parasites.
 106. The method ofclaim 105, wherein said viral pathogen is selected from the groupconsisting of hepatitis type A, hepatitis type B, hepatitis type C,influenza, varicella, adenovirus, herpes simplex type I (HSV-I), herpessimplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus,respiratory syncytial virus (RSV), papilloma virus, papova virus,cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus,mumps virus, measles virus, rubella virus, polio virus, humanimmunodeficiency virus type I (HIV-I), and human immunodeficiency virustype II (HIV-II).
 107. The method of claim 105, wherein said bacterialpathogen is selected from the group consisting of Mycobacteria,Mycoplasma, Neisseria, and Legionella.
 108. The method of claim 105,wherein said protozoal pathogen is selected from the group consisting ofLeishmania, Kokzidioa, Trypanosoma, Chlamydia, and Rickettsia.
 109. Aproduct produced by the method of claim
 93. 110. A method for preparingan immunogenic composition for inducing an immune response in avertebrate subject, the method comprising: (a) reconstituting in vitroan antigenic molecule and a Hsp90 protein molecule in the presence of amodulator of the biological activity of a Hsp90 protein to therebyproduce an immunogenic complex comprising a Hsp90 protein non-covalentlycovalently bound to an antigenic molecule, said complex, whenadministered to said vertebrate subject being operative at initiating animmune response in said vertebrate subject; and (b) combining saidcomplex with a pharmaceutically acceptable carrier.
 111. The method ofclaim 110, wherein the Hsp90 protein is GRP94 or HSP90.
 112. The methodof claim 110, wherein the antigenic molecule is a peptide with which theHsp90 protein is endogenously associated in vivo.
 113. The method ofclaim 110, wherein the antigenic molecule is an exogenous antigenicpeptide.
 114. The method of claim 110, wherein the antigenic molecule isa cancer antigen.
 115. The method of claim 114, wherein the cancerantigen is from a cancer comprising a sarcoma or carcinoma, selectedfrom the group consisting of fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma, leukemia, lymphoma, multiple myeloma, Waldenströom'smacroglobulinemia, and heavy chain disease.
 116. The method of claim110, wherein the antigen molecule is peptide from a pathogen.
 117. Themethod of claim 116, wherein said pathogen is selected from the groupconsisting of viruses, bacteria, fungi, protozoa, and parasites. 118.The method of claim 117, wherein said viral pathogen is selected fromthe group consisting of hepatitis type A, hepatitis type B, hepatitistype C, influenza, varicella, adenovirus, herpes simplex type I (HSV-I),herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus,rotavirus, respiratory syncytial virus (RSV), papilloma virus, papovavirus, cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackievirus, mumps virus, measles virus, rubella virus, polio virus, humanimmunodeficiency virus type I (HIV-I), and human immunodeficiency virustype II (HIV-II).
 119. The method of claim 117, wherein said bacterialpathogen is selected from the group consisting of Mycobacteria,Mycoplasma, Neisseria, and Legionella.
 120. The method of claim 117,wherein said protozoal pathogen is selected from the group consisting ofLeishmania, Kokzidioa, Trypanosoma, Chlamydia, and Rickettsia.
 121. Aproduct produced by the method of claim
 110. 122. A method for preparingan immunogenic composition for inducing an immune response in avertebrate subject, the method comprising: (a) sensitizing one or moreantigen presenting cells in vitro with a complex comprising a Hsp90protein non-covalently bound to an antigenic molecule and with anactivating ligand of a Hsp90 protein; and (b) combining said one or moresensitized antigen presenting cell with pharmaceutically acceptablecarrier.
 123. The method of claim 122, wherein the Hsp90 protein isGRP94 or HSP90.
 124. The method of claim 122, wherein the antigenicmolecule is a peptide with which the Hsp90 protein is endogenouslyassociated in vivo.
 125. The method of claim 122, wherein the antigenicmolecule is an exogenous antigenic peptide.
 126. The method of claim122, wherein the antigenic molecule is a cancer antigen.
 127. The methodof claim 126, wherein the cancer antigen is from a cancer comprising asarcoma or carcinoma, selected from the group consisting offibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma, leukemia, lymphoma, multiple myeloma, Waldenströom'smacroglobulinemia, and heavy chain disease.
 128. The method of claim122, wherein the antigen molecule is a peptide from a pathogen.
 129. Themethod of claim 128, wherein said pathogen is selected from the groupconsisting of viruses, bacteria, fungi, protozoa, and parasites. 130.The method of claim 129, wherein said viral pathogen is selected fromthe group consisting of hepatitis type A, hepatitis type B, hepatitistype C, influenza, varicella, adenovirus, herpes simplex type I (HSV-I),herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus,rotavirus, respiratory syncytial virus (RSV), papilloma virus, papovavirus, cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackievirus, mumps virus, measles virus, rubella virus, polio virus, humanimmunodeficiency virus type I (HIV-I), and human immunodeficiency virustype II (HIV-II).
 131. The method of claim 129, wherein said bacterialpathogen is selected from the group consisting of Mycobacteria,Mycoplasma, Neisseria, and Legionella.
 132. The method of claim 129,wherein said protozoal pathogen is selected from the group consisting ofLeishmania, Kokzidioa, Trypanosoma, Chlamydia, and Rickettsia.
 133. Aproduct produced by the method of claim 122.